Modifications of HIV Env, Gag, and Pol enhance immunogenicity for genetic immunization

ABSTRACT

Modified HIV Env, Gag, Pol, or Nef DNA with improved ability to elicit antibody and CTL responses to HIV antigens have been identified as prototype immunogens for the treatment and prevention of HIV infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/359,120, filed Feb. 4, 2003, which is a continuation of internationalapplication number PCT/US01/25721, and claims the benefit of priority ofinternational application number PCT/US01/25721 having an internationalfiling date of Aug. 14, 2001, designating the United States of Americaand published in English, which claims the benefit of priority of U.S.provisional patent application No. 60/279,257, filed Mar. 28, 2001, U.S.provisional patent application No. 60/252,115, filed Nov. 14, 2000, andU.S. provisional patent application No. 60/225,097, filed Aug. 14, 2000;all of which are hereby expressly incorporated by reference in theirentireties.

SEQUENCE LISTING

The present application is being filed along with duplicate copies of aCD-ROM marked “Copy 1” and “Copy 2” containing a Sequence Listing inelectronic format. The duplicate copies of the CD-ROM each contain afile entitled “NIH206.001 CDV1” created on Nov. 10, 2008 which is1,528,044 bytes in size. The information on these duplicate CD-ROMs isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology. Thepresent invention discloses modified HIV Env, Gag, Pol, and Nefproteins, related nucleotide sequences, and usage for geneticimmunization.

BACKGROUND OF THE INVENTION

Protective immunity against human immunodeficiency virus-1 (HIV-1) islikely to require recognition of linear and conformation epitopes frommultiple HIV antigens. Whether these responses can be elicited moreeffectively by virion-like structures or fused CTL epitopes is unknown.

The immune response to HIV infection in long-term non-progressors andHIV-exposed sex workers suggests that specific viral immunity may limitinfection and the symptoms of disease. No single characteristic yetcorrelates with protective immunity, but studies in non-human ofprimates suggest that both humoral and cellular immunity are requiredfor this response. Depletion of cytotoxic T cells (CTLs) inchronically-infected macaques enhances viremia. In humans, higher CTLresponses correlate with lower viral load and stabilization of clinicalsymptoms. In animal models, passive transfer of neutralizing antibodiescan also contribute to protection against virus challenge. Neutralizingantibody responses can also be developed in HIV-infected individuals andare associated with lower viral loads in long-term non-progressors.Though this neutralizing antibody response is uncommon, it is directedlargely against the Env protein of the virus.

In early human vaccine trials, gp120 protein immunogens have yieldeddisappointing results: vaccine-induced antibodies have not been broadlyneutralizing and have sometimes enhanced infection in vitro. Monomericgp120 loses oligomer-dependent epitopes and does not include sequencesin the ectodomain of the gp41 that become exposed during virus entry. Itis assumed that broadly neutralizing antibodies bind to nativegp120/gp41 complex on the surface of the virus rather than solublegp120.

The development of a cytotoxic T lymphocyte (CTL) response to viruses isoften crucial to the outcome of infections. Lysis of infected cellsprior to the production of progeny virions may limit virus burst size(Yang, O et al., 1996, J. Virol., 70:5799-5806), and HIV specific CD8⁺cytotoxic T lymphocytes (CTL) have been shown to be important in viralclearance and in the control of initial HIV-1 spread (Borrow, P et al.,1994, J. Virol., 68:6103-6110; Yang, O et al., 1996, J. Virol.70:5799-5806). CTL responses specific to HIV also contribute toreduction in viral load during acute and asymptomatic infection (Klein,M R, et al., 1995, J. Exp. Med. 181:1365-1372; Moss, P A H et al., 1995,Proc. Natl. Acad. Sci. USA, 92:5773-5777) and may be involved inprotection against the establishment of persistent HIV infections(Rowland-Jones, S L et al., 1993, Lancet, 341:860-861; Rowland-Jones, SL et. al., 1995, Nat. Med., 1:59-64). High-frequency CTL responses toHIV-1 correlated with low viral load and slow disease progression inchronically infected individuals (Musey, L et al., 1997, N. Engl. J.Med., 337:1267-1274; Ogg, G S et al., 1998, Science, 279:2103-2106.).More compelling evidence of an antiviral effect of CD8⁺ cells wasdemonstrated in controlled studies in macaques, in which CD8⁺ cells weredepleted in vivo using a monoclonal antibody. The viral loads in theseanimals increased or decreased as the CD8⁺ cells were depleted orreappeared, respectively (Jin, X et al., 1999, J. Exp. Med.,189:991-998; Schmitz, J E, et al., 1999, Science, 283:857-860).Therefore, induction of a CTL response specific to these proteinsrepresents a desirable response in an HIV-1 vaccine.

HIV-1 internal structural and enzymatic proteins contain conserveddomains that preserve their functions and thus exhibit less antigenicdiversity that may elicit more effective CTL responses (Nixon, D F etal., 1988, Nature, 336:484-487.). Efficient and durable CTL responsesrequire endogenous antigen synthesis and processing. Current vaccinedelivery techniques include immunization with live, attenuated viruses,inactivated recombinant virus infection (Letvin, N L, 1998, Science,280:1875-1880) or plasmid DNA expression vectors. A major obstacle inthe induction of CTL responses with naked DNA or recombinant virusduring development of an HIV vaccine is that the expression of HIV-1structural and enzymatic genes is tightly regulated by the virus itself.The expression of these proteins is heavily dependent upon the existenceof the Rev-responsive element (RRE) of HIV-1 in recombinant vectors(Cullen, B R, 1992, Microbiol. Rev., 56:375-394; Felber, B K et al.,1989, Proc Natl Acad Sci USA, 86:1495-1499). Poor expression is causedby the presence of AT rich inhibitory nucleotide sequences (INS) in thegag, pol and env genes, which inhibit the nuclear export and efficientexpression of unspliced HIV1 mRNAs. Early studies of DNA vaccinationagainst HIV in mice required the inclusion of Rev in their expressionvectors (Lu, S et al., 1995, Virology, 209:147-154; Okuda, K et al.,1995, AIDS Res. Hum. Retroviruses, 11:933-943; Wang, B et al., 1993,Proc. Natl. Acad. Sci. U.S. A 90:4156-4160), but modification of INS hasbeen shown to facilitate Rev-independent expression of HIV-1 Gag (Qiu,J-T et al., 1999, J. Virol., 73:9145-9152; zur Megede, J et al., 2000, JVirol 74:2628-2635), allowing detectable humoral and CTL responsesagainst this protein (Qiu, J-T et al., 1999, J. Virol., 73:9145-9152).These modified HIV-1 Gag genes produced viral-like particles of theexpected density and morphology and induced an immune response to HIV-1Gag after DNA immunization in mice (zur Megede, J et al., 2000, J Virol,74:2628-2635).

SUMMARY OF THE INVENTION

Protective immunity against human immunodeficiency virus-1 (HIV-1) islikely to require recognition of linear and conformation epitopes frommultiple HIV antigens, and whether these responses can be elicited moreeffectively by virion-like structures or fused CTL epitopes waspreviously unknown. Herein is provided a modified HIV Env with deletionsin the cleavage site, fusogenic domain, and spacing of heptad repeats 1and 2 to expose the core protein for optimal antigen presentation andrecognition. Additionally we provide a Gag-Pol or Gag-Pol-Nef fusionprotein that is a polyprotein designed to maximize epitope presentation.The invention extends the mutation in HIV Env, Gag, Pol, and Nef to anyHIV clade or strain and to related proteins of other viruses. Differentcombinations, different orders, and different variations on a theme areenvisioned, the theme being to optimize presentation of epitopes thatgenerate broad CTL and antibody responses.

More particularly, we have investigated the effect of specific mutationsin human immunodeficiency virus type 1 (HIV-1) envelope (Env) on humoraland cellular immune responses after DNA vaccination. Mice were injectedwith plasmid expression vectors encoding HIV Env with modifications ofconserved glycosylation sites or different COOH-terminal mutationsintended to mimic a fusion intermediate. Elimination of conservedglycosylation sites did not substantially enhance humoral or CTLimmunity. In contrast, a modified gp140 with deletions in the cleavagesite, fusogenic domain and spacing of heptad repeats 1 and 2 enhancedhumoral immunity without reducing the efficacy of the CTL response.Because of its ability to stimulate the antibody response to nativegp160 without affecting cellular immunity, this modified gp140 or arelated derivative is envisioned to be a useful component of an AIDSvaccine.

In addition, we have examined the immune response to HIV-1 Gag and Polafter plasmid DNA immunization with Rev-independent expression vectorsencoding various forms of these proteins. Immune responses were analyzedafter vaccination with four expression vectors, including Gag alone orGag-Pol, both of which gave rise to virion-like particles (VLPs),compared to Pol alone or a Gag-Pol fusion protein that did not formVLPs. The Gag-Pol fusion protein induced the most broad and potent CTLresponses to Gag and Pol in DNA-vaccinated mice, and this immunogen alsoreadily elicited an antibody response to HIV-1 Gag and Pol determinants.Through its ability to induce broad CTL and antibody responses, thisGag-Pol fusion protein or a related derivative is envisioned to be auseful component of an AIDS vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1-176. Plasmid Maps. Table 1 provides the description of FIGS.1-176, each of which illustrates a map of a plasmid described herein.

FIG. 1. Plasmid 2100.

FIG. 2. Plasmid 2200.

FIG. 3. Plasmid 2300.

FIG. 4. Plasmid 2302.

FIG. 5. Plasmid 2400.

FIG. 6. Plasmid 2700.

FIG. 7. Plasmid 2701.

FIG. 8. Plasmid 2702.

FIG. 9. Plasmid 2706.

FIG. 10. Plasmid 2707.

FIG. 11. Plasmid 2800.

FIG. 12. Plasmid 2801.

FIG. 13. Plasmid 2804.

FIG. 14. Plasmid 2805.

FIG. 15. Plasmid 2810.

FIG. 16. Plasmid 2811.

FIG. 17. Plasmid 2812.

FIG. 18. Plasmid 2813.

FIG. 19. Plasmid 2814.

FIG. 20. Plasmid 2820.

FIG. 21. Plasmid 2821.

FIG. 22. Plasmid 2822.

FIG. 23. Plasmid 2823.

FIG. 24. Plasmid 2824.

FIG. 25. Plasmid 2825.

FIG. 26. Plasmid 2826.

FIG. 27. Plasmid 2827.

FIG. 28. Plasmid 2828.

FIG. 29. Plasmid 2829.

FIG. 30. Plasmid 2830.

FIG. 31. Plasmid 2831.

FIG. 32. Plasmid 2832.

FIG. 33. Plasmid 2833.

FIG. 34. Plasmid 2834.

FIG. 35. Plasmid 2835.

FIG. 36. Plasmid 2836.

FIG. 37. Plasmid 2837.

FIG. 38. Plasmid 2838.

FIG. 39. Plasmid 2839.

FIG. 40. Plasmid 2840.

FIG. 41. Plasmid 2841.

FIG. 42. Plasmid 2842.

FIG. 43. Plasmid 2843.

FIG. 44. Plasmid 2844.

FIG. 45. Plasmid 2845.

FIG. 46. Plasmid 2846.

FIG. 47. Plasmid 2847.

FIG. 48. Plasmid 2848.

FIG. 49. Plasmid 2849.

FIG. 50. Plasmid 2850.

FIG. 51. Plasmid 2851.

FIG. 52. Plasmid 2852.

FIG. 53. Plasmid 2853.

FIG. 54. Plasmid 2854.

FIG. 55. Plasmid 2860.

FIG. 56. Plasmid 2861.

FIG. 57. Plasmid 2862.

FIG. 58. Plasmid 2863.

FIG. 59. Plasmid 2864.

FIG. 60. Plasmid 2865.

FIG. 61. Plasmid 2866.

FIG. 62. Plasmid 2867.

FIG. 63. Plasmid 2868.

FIG. 64. Plasmid 2869.

FIG. 65. Plasmid 2870.

FIG. 66. Plasmid 2871.

FIG. 67. Plasmid 2872.

FIG. 68. Plasmid 2873.

FIG. 69. Plasmid 2874.

FIG. 70. Plasmid 2900.

FIG. 71. Plasmid 3000.

FIG. 72. Plasmid 3200.

FIG. 73. Plasmid 3201.

FIG. 74. Plasmid 3202.

FIG. 75. Plasmid 3203.

FIG. 76. Plasmid 3300.

FIG. 77. Plasmid 3301.

FIG. 78. Plasmid 3400.

FIG. 79. Plasmid 3401.

FIG. 80. Plasmid 3500.

FIG. 81. Plasmid 3600.

FIG. 82. Plasmid 3700.

FIG. 83. Plasmid 3800.

FIG. 84. Plasmid 5200.

FIG. 85. Plasmid 5201.

FIG. 86. Plasmid 5202.

FIG. 87. Plasmid 5203.

FIG. 88. Plasmid 5300.

FIG. 89. Plasmid 5301.

FIG. 90. Plasmid 5303.

FIG. 91. Plasmid 5304.

FIG. 92. Plasmid 5305.

FIG. 93. Plasmid 5306.

FIG. 94. Plasmid 5307.

FIG. 95. Plasmid 5308.

FIG. 96. Plasmid 5309.

FIG. 97. Plasmid 5350.

FIG. 98. Plasmid 5351.

FIG. 99. Plasmid 5352.

FIG. 100. Plasmid 5353.

FIG. 101. Plasmid 5354.

FIG. 102. Plasmid 5355.

FIG. 103. Plasmid 5356.

FIG. 104. Plasmid 5357.

FIG. 105. Plasmid 5358.

FIG. 106. Plasmid 5359.

FIG. 107. Plasmid 5360.

FIG. 108. Plasmid 5361.

FIG. 109. Plasmid 5362.

FIG. 110. Plasmid 5363.

FIG. 111. Plasmid 5364.

FIG. 112. Plasmid 5365.

FIG. 113. Plasmid 5366.

FIG. 114. Plasmid 5367.

FIG. 115. Plasmid 5368.

FIG. 116. Plasmid 5369.

FIG. 117. Plasmid 5370.

FIG. 118. Plasmid 5371.

FIG. 119. Plasmid 5372.

FIG. 120. Plasmid 5373.

FIG. 121. Plasmid 5374.

FIG. 122. Plasmid 5375.

FIG. 123. Plasmid 5376.

FIG. 124. Plasmid 5377.

FIG. 125. Plasmid 5378.

FIG. 126. Plasmid 5379.

FIG. 127. Plasmid 5500.

FIG. 128. Plasmid 5501.

FIG. 129. Plasmid 5502.

FIG. 130. Plasmid 5503.

FIG. 131. Plasmid 5504.

FIG. 132. Plasmid 5505.

FIG. 133. Plasmid 5506.

FIG. 134. Plasmid 5507.

FIG. 135. Plasmid 5508.

FIG. 136. Plasmid 5509.

FIG. 137. Plasmid 5510.

FIG. 138. Plasmid 5511.

FIG. 139. Plasmid 5512.

FIG. 140. Plasmid 5513.

FIG. 141. Plasmid 5514.

FIG. 142. Plasmid 5515.

FIG. 143. Plasmid 5516.

FIG. 144. Plasmid 5517.

FIG. 145. Plasmid 5518.

FIG. 146. Plasmid 5519.

FIG. 147. Plasmid 5520.

FIG. 148. Plasmid 5521.

FIG. 149. Plasmid 5522.

FIG. 150. Plasmid 5523.

FIG. 151. Plasmid 5524.

FIG. 152. Plasmid 5525.

FIG. 153. Plasmid 5526.

FIG. 154. Plasmid 5527.

FIG. 155. Plasmid 5528.

FIG. 156. Plasmid 5529.

FIG. 157. Plasmid 3900.

FIG. 158. Plasmid 3901.

FIG. 159. Plasmid 4000.

FIG. 160. Plasmid 4001.

FIG. 161. Plasmid 4100.

FIG. 162. Plasmid 4101.

FIG. 163. Plasmid 4200.

FIG. 164. Plasmid 4300.

FIG. 165. Plasmid 4301.

FIG. 166. Plasmid 4302.

FIG. 167. Plasmid 4303.

FIG. 168. Plasmid 4304.

FIG. 169. Plasmid 4305.

FIG. 170. Plasmid 4306.

FIG. 171. Plasmid 4308.

FIG. 172. Plasmid 4309.

FIG. 173. Plasmid 4310.

FIG. 174. Plasmid 4311.

FIG. 175. Plasmid 4312.

FIG. 176. Plasmid 4313.

FIG. 177. Comparison of GP2 with the structures of viral and cellularmembrane fusion proteins. (A) Recombinant Ebola Zaire GP2, (B)Recombinant Mo-55 from the TM subunit of MoMuLv, (C) Low pH-treated HA2from influenza virus, (D) Recombinant, proteolysis-resistant core ofHIV-1 gp41, (E) Recombinant SIV gp41, NMR structure, (F) Recombinantcore coiled segments of the SNARES syntaxin 1-A, synaptobrevin-II, andSNAP-25B. Weissenhorn et al., 1998, Molecular Cell, 2, 605-616.

FIG. 178. Schematic representation of functional domains and mutationsin HIV-1 Env glycoproteins. Full-length envelope polyprotein, gp160,with the indicated features based on the amino acid residues of HXB2 isshown (top). Functional domains include the gp120/gp41 cleavage site(residues 510/511), the fusion domain (512-527), the two heptad repeats(546-579 and 628-655), the transmembrane domain (684-705), and thecytoplasmic domain (706-856). The mutant forms of the envelope proteinsare shown below the structure of gp160. COOH deletions were introducedthat terminate the envelope protein at positions 752, 704, or 680 toproduce gp150, gp145, or gp140, respectively. Two internal deletionsthat removed the cleavage site, the fusion domain, and the regionbetween the two heptad repeats were introduced into gp160, gp150, gp145,and gp140. A further deletion in the COOH-terminal region at position592 removed the second heptad repeat, the transmembrane domain, and theinterspace region to produce gp128ΔCFI. To disrupt potentialglycosylation sites, asparagine (N) residues at eleven positions (88,156, 160, 197, 230, 234, 241, 262, 276, 289, and 295) were replaced withaspartic acid (D) residues in both gp160 and gp150. Versions of bothgp160 and gp150 were created with a total of 17 mutated glycosylationsites by including six additional N to D substitutions at positions 332,339, 356, 386, 392, and 448.

FIG. 179. Comparison of the expression of the HIV-1 gp160 withcodon-optimized gp160. A. Expression of plasmids encoding Rev-dependentand Rev-independent codon-modified gp160. Upper panel: expression ofRev-dependent viral gp160 (left) and codon-modified gp160 (right) intransfected 293 cells. Lower panel: comparable expression of α-actin inthese transfected cells. B. Expression of mutant CXCR4-tropic HIV Envglycoproteins with COOH-terminal truncations. C and D, respectively.CXCR4-tropic envelope proteins containing mutant glycosylation sites andmutant functional domains. The indicated proteins were detected byimmunoblotting as above. Cell lysates produced by transfection withvector containing no insert were used as controls (first lane in eachpanel).

FIG. 180. Cytotoxicity of full-length gp160 is eliminated by deletion ofthe COOH-terminal cytoplasmic domain. Cell rounding and detachment wasnot observed in control-transfected 293 cells (A), in contrast tofull-length gp160 (B) and to a lesser extent in cells transfected withgp150 (C), in contrast to gp145 (D) or gp140 (E).

FIG. 181. Expression of soluble gp140ΔCFI HIV-1 envelope variant.Immunoprecipitation and Western blot analysis of supernatants from theindicated transfected cells.

FIG. 182. Antibody response against HIV-1 envelope proteins in DNAimmunized mice. A. Comparison of the antibody response in mice immunizedwith gp140 (ΔCFI) or other Env plasmid expression vectors. Sera werecollected 2 weeks after the last immunization and used toimmunoprecipitate codon-altered gp160 from lysates of transfected 293cells. The quantitation of the immunoprecipitated gp160 was done asdescribed in FIG. 182B. The average of the normalized data has beenpresented as a bar diagram. B. Antibody responses in mice immunized withdifferent mutant Env expression vectors. Antisera from immunized micewere diluted in IP buffer and 1 μl of each diluted serum was used toimmunoprecipitate codon-altered HIV-1 gp160 from lysates of transfected293 cells as described in FIG. 180A. The gels were scanned and theintensity of the gp160 band was determined by densitometry using theprogram Image Quant and presented relative to the intensity of gp160immunoprecipitated with positive control sera (rabbit anti-gp160), whichwas used to normalize data between experiments. These data are presentedgraphically to facilitate comparison among groups. C. Antibody responsesin mice immunized with gp140 or gp140 (ΔCFI) were determined byimmunoprecipitation and Western blotting. Animals received two boosterdoses (100 μg) of the same plasmid, two weeks apart. Sera (1 μl)collected 2 weeks after the last immunization was used toimmunoprecipitate codon-optimized HIV-1 gp160 from lysates oftransfected 293 cells containing 400 μg of total protein. Each lanecorresponds to the sera from an animal immunized with either the controlvector (lanes 1 and 2), CXCR4-tropic gp140 (lanes 3-6), or plasmid thatexpresses gp140 with the indicated mutant functional domains (lanes7-10). A mouse monoclonal antibody to gp160 (HIV-1 V3 Monoclonal(IIIB-V3-13), NIH AIDS Reagent Program) was used as a positive control(lane 11).

FIG. 183. CTL response against HIV-1 envelope proteins in DNA immunizedmice. The CTL response to CXCR4-tropic Env and indicated deletionmutants is shown (A). Dependence of CTL activity on CD8 cells was shownby magnetic bead depletion using the indicated representative immunogens(B). The CTL responses to CXCR4-tropic envelope with glycosylation siteand ΔCFI mutations are shown (C and D, respectively). Spleen cells wereisolated from immunized mice two weeks after the final immunization andstimulated in vitro with irradiated cells expressing gp160 with additionof hIL2 (5 U/ml) at day 4. The cytolytic activity of the restimulatedspleen cells was tested after 7 days against V3 peptide-pulsed BC10MEcells. Similar findings were observed with target cells that stablyexpress full-length Env.

FIG. 184. Schematic representation of HIV-1 Gag-Pol expressionconstructs. The protein sequences of Gag (amino acids 1-432) from HXB2(GenBank accession number K03455) and Pol (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) were used to create a syntheticversion of hGag-Pol using codons found in human cells. hGag-PolΔFSΔPrwas made by modification of the frame shift site (FS) and inactivationof protease. For hPol, 432 amino acids were deleted from theNH₂-terminal region of hGag-Pol and addition of an ATG codon. hGag wasmade by deletion of 925 amino acids from the COOH-terminal region ofhGag-Pol. hGag-Pol, hGag-PolΔFSΔPr, hPol and hGag are expressed from thepNGVL-3 vector backbone.

FIG. 185. HIV-1 Gag-Pol expression in transfected 293T cells and stablytransfected CT26 and BC10 ME cells. Cell lysates from 293T cellstransfected with pCMV ΔR8.2 containing viral Gag-Pol (vGag-Pol),pNGVL-hGag, hPol, hGag-PolΔFSΔPr and hGag-Pol were separated by 4-15%gradient SDS-PAGE, transferred to nitrocellulose filters, and analyzedby immunoblotting with (A) human anti HIV-1-IgG, (B) monoclonalanti-p24, and (C) rabbit anti-RT. (D) Cell lysates from CT26 and BC10MEcells stably transduced with either hGag or hPol were analyzed withhuman anti HIV-1-IgG.

FIG. 186. Transmission electron microscopy of HIV-1 immature virus-likeparticles (VLP) produced by transfected 293T. Cells were transfectedwith pNGVL-hGag 48 hours prior to harvesting and fixing (magnification25,000×).

FIG. 187. Gag or Pol specific CTL response mediated by CD8 positivecells in immunized mice. Two weeks after mice were immunized with acontrol vector, hGag, hPol, hGag-PolΔFSΔPr, and hGag-Pol, splenic cellswere harvested and sensitized with naïve mouse splenic cells pulsed withGag or Pol peptides. One week later, effector cells were tested forcytolytic activity in a 5-h ⁵¹Cr release assay using ⁵¹Cr-labeled BC10MEtarget cells that were pulsed for 2 hours with either (A) HIV-1 Gagpeptides, or (B) HIV-1 Pol peptides. (C) CD4+ or CD8+ lymphocytes weredepleted from splenic cells of immunized mice with anti-mouse-CD4+ orCD8+ Dynal beads according to the manufacturer's instructions.

FIG. 188. Gag or Pol specific CTL response mediated by CD8 positivecells in immunized mice using stable expressing cell lines as targetcells. Two weeks after immunization in mice, splenic cells wereharvested and sensitized with naïve mouse splenic cells pulsed with Gagor Pol peptides. One week later, effector cells were tested forcytolytic activity in a 5-h ⁵¹Cr release assay using ⁵¹Cr-labeled BC10MEtarget cells expressing either (A) HIV-1 Gag or (B) Pol protein.

FIG. 189. HIV-1 p24 antibody ELISA assays, HIV-1 immunoblotting andimmunoprecipitation Western blotting. (A) An HIV-1 p24 antibody ELISAassay was performed by coating 96-well plates with 50 μl of purifiedrecombinant HIV-1_(IIIB) p24 antigen at a concentration of 2 μg/ml inPBS buffer, pH 7.4. (B) HIV-1 immunoblotting of strips containing HIV-1proteins were incubated with pooled mouse sera at a dilution of 1:25.Bands were visualized using the ECL western blotting detection reagent.(C) Immunoprecipitation and Western blotting of hPol gene-transfected293T cell lysates three days after transfection with RIPA buffer. Thepooled mouse serum was diluted with IP buffer. After adding 10 μg of thecell lysate containing HIV-1 Pol protein, the reactions were incubatedovernight on a rotator at 4° C. The next day, 250 μl of Protein G and ASepharose beads (10% V/V in IP buffer) were added, and the reactionswere incubated on a rotator for 2 hours at 4° C. The reactions werewashed 4× with IP buffer, re-suspended with 30 μl of 1× sample buffer,and then loaded onto SDS-PAGE. The reactions were transferred to anImmobilon P membrane, and then incubated with anti HIV-1-IgG. Bands werevisualized using the ECL Western blotting detection reagent.

TABLE 1 SEQ ID Plasmid Plasmid Name/Description Plasmid Map Name NO FIG.Env Plasmids 2100 pVR1012x/s R5gp139-Nef (delta) MHC (delta) CD4/hpVR1012x/s R5gp139-Nef delta MHC delta CD4/h 1 1 2200 pVR1012x/sR5gp157-Nef (delta) MHC (delta) CD4/h pVR1012x/s R5gp157-Nef deltaMHCdeltaCD4/h 2 2 2300 pVR1012x/s X4 gp139-Nef deltaMHC deltaCD4/hpVR1012x/s X4gp139-Nef delta MHC delta CD4/h 3 3 2302 pVR1012x/sX4gp130-Nef/h pVR1012x/s X4gp130-Nef/h 4 4 2400 pVR1012x/sX4gp157-NefDMHCDCD4/h pVR1012x/sX4gp157-Nef delta MHC delta CD4/h 5 52700 pVR1012x/s X4gp140/h pVR1012x/s X4gp140/h 6 6 2701 pVR1012x/sX4gp140 ΔCFI/h OR pVR1012x/s X4gp140 (del pVR1012x/s X4gp140 (del F/CLdel H IS)/h 7 7 F/CL del H IS)/h 2702 pVR1012x/s X4gp128 ΔCFI/h ORpVR1012x/s X4gp128 (del pVR1012x/s X4gp128 (del F/CL)/h 8 8 F/CL)/h 2706pVR1012x/s X4gp145/h pVR1012x/s X4gp145/h 9 9 2707 pVR1012x/s X4gp145ΔCFI/h OR pVR1012x/s X4gp145 (del pVR1012x/s X4gp145 (del F/CL del HIS)/h 10 10 F/CL del H IS)/h 2800 pVR1012x/s R5gp140/h pVR1012x/sR5gp140/h 11 11 2801 pVR1012x/s R5gp140 ΔCFI/h OR pVR1012x/sR5gp140 (delpVR1012x/sR5gp140 (del F/CL del H IS)/h 12 12 F/CL del H IS)/h 2804pVR1012x/s R5gp145/h pVR1012x/s R5gp145/h 13 13 2805 pVR1012x/s R5gp145ΔCFI/h OR pVR1012x/s R5gp145 (del pVR1012x/sR5gp145 (del F/CL del HIS)/h 14 14 F/CL del H IS)/h 2810 pVR1012x/s R5gp140delC1 (delCFI)/hpVR1012x/sR5gp140delC1 (delCFI)/h 15 15 2811 pVR1012x/s R5gp140delC2(delCFI)/h pVR1012x/sR5gp140delC2 (delCFI)/h 16 16 2812 pVR1012x/sR5gp140delC3 (delCFI)/h pVR1012x/sR5gp140delC3 (delCFI)/h 17 17 2813pVR1012x/s R5gp140delC4 (delCFI)/h pVR1012x/sR5gp140delC4 (delCFI)/h 1818 2814 pVR1012x/s R5gp140delC5 (delCFI)/h pVR1012x/sR5gp140delC5(delCFI)/h 19 19 2820 pVR1012x/s R5gp140 (dCFI)/dV1 pVR1012x/s R5gp140(dCFI) dV1/h 20 20 2821 pVR1012x/s R5gp140 (dCFI)/dV2 pVR1012x/s gp140(dCFI) dV2/h 21 21 2822 pVR1012x/s R5gp140 (dCFI)/dV3 pVR1012x/s gp140(dCFI) dV3/h 22 22 2823 pVR1012x/s R5gp140 (dCFI)/dV4 pVR1012x/s R5gp140(dCFI) dV4/h 23 23 2824 pVR1012x/s R5gp140 (dCFI)/dV12 pVR1012x/sR5gp140 (dCFI) dV12/h 24 24 2825 pVR1012x/s R5gp140 (dCFI)/dV13pVR1012x/s R5gp140 (dCFI) dV13/h 25 25 2826 pVR1012x/s R5gp140(dCFI)/dV14 pVR1012x/s R5gp140 (dCFI) dV14/h 26 26 2827 pVR1012x/sR5gp140 (dCFI)/dv23 pVR1012x/s R5gp140 (dCFI) dv23/h 27 27 2828pVR1012x/s R5gp140 (dCFI)/dv24 pVR1012x/s R5gp140 (dCFI) dv24/h 28 282829 pVR1012x/s R5gp140 (dCFI)/dv34 pVR1012x/s R5gp140 (dCFI) dv34/h 2929 2830 pVR1012x/s R5gp140 (dCFI)/dv123 pVR1012x/s R5gp140 (dCFI)dv123/h 30 30 2831 pVR1012x/s R5gp140 (dCFI)/dv124 pVR1012x/s R5gp140(dCFI) dv124/h 31 31 2832 pVR1012x/s R5gp140 (dCFI)/dv134 pVR1012x/sR5gp140 (dCFI) dv134/h 32 32 2833 pVR1012x/s R5gp140 (dCFI)/dv234pVR1012x/s R5gp140 (dCFI) dv234/h 33 33 2834 pVR1012x/s R5gp140(dCFI)/dV1234 pVR1012x/s R5gp140 (dCFI) dv1234/h 34 34 2835 pAdAptR5gp140 (dCFI)/dV1 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv1/h 35 35 2836pAdApt R5gp140 (dCFI)/dV2 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv2/h 36 362837 pAdApt R5gp140 (dCFI)/dV3 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv3/h37 37 2838 pAdApt R5gp140 (dCFI)/dV4 pAdApt CMV TbGH (+) R5gp140 (dCFI)dv4/h 38 38 2839 pAdApt R5gp140 (dCFI)/dV12 pAdApt CMV TbGH (+) R5gp140(dCFI) dv12/h 39 39 2840 pAdApt R5gp140 (dCFI)/dV13 pAdApt CMV TbGH (+)R5gp140 (dCFI) dv13/h 40 40 2841 pAdApt R5gp140 (dCFI)/dV14 pAdApt CMVTbGH (+) R5gp140 (dCFI) dv14/h 41 41 2842 pAdApt R5gp140 (dCFI)/dV23pAdApt CMV TbGH (+) R5gp140 (dCFI) dv23/h 42 42 2843 pAdApt R5gp140(dCFI)/dV24 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv24/h 43 43 2844 pAdAptR5gp140 (dCFI)/dV34 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv34/h 44 44 2845pAdApt R5gp140 (dCFI)/dV123 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv123/h45 45 2846 pAdApt R5gp140 (dCFI)/dV124 pAdApt CMV TbGH (+) R5gp140(dCFI) dv124/h 46 46 2847 pAdApt R5gp140 (dCFI)/dV134 pAdApt CMV TbGH(+) R5gp140 (dCFI) dv134/h 47 47 2848 pAdApt R5gp140 (dCFI)/dV234 pAdAptCMV TbGH (+) R5gp140 (dCFI) dv234/h 48 48 2849 pAdApt R5gp140(dCFI)/dV1234 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv1234/h 49 49 2850pVR1012x/s R5gp145delC1 (delCFI)/h pVR1012x/sR5gp145delC1 (delCFI)/h 5050 2851 pVR1012x/s R5gp145delC2 (delCFI)/h pVR1012x/sR5gp145delC2(delCFI)/h 51 51 2852 pVR1012x/s R5gp145delC3 (delCFI)/hpVR1012x/sR5gp145delC3 (delCFI)/h 52 52 2853 pVR1012x/s R5gp145delC4(delCFI)/h pVR1012x/sR5gp145delC4 (delCFI)/h 53 53 2854 pVR1012x/sR5gp145delC5 (delCFI)/h pVR1012x/sR5gp145delC5 (delCFI)/h 54 54 2860pVR1012x/s R5gp145 (dCFI)/h/dV1 pVR1012x/s R5 gp145 (dCFI) dv1/h 55 552861 pVR1012x/s R5gp145 (dCFI)/h/dV2 pVR1012x/s R5gp145 (dCFI) dv2/h 5656 2862 pVR1012x/s R5gp145 (dCFI)/h/dV3 pVR1012x/s R5gp145 (dCFI) dv3/h57 57 2863 pVR1012x/s R5gp145 (dCFI)/h/dV4 pVR1012x/s R5gp145 (dCFI)dv4/h 58 58 2864 pVR1012x/s R5gp145 (dCFI)/h/dV12 pVR1012x/s R5gp145(dCFI) dv12/h 59 59 2865 pVR1012x/s R5gp145 (dCFI)/h/dV13 pVR1012x/sR5gp145 (dCFI) dv13/h 60 60 2866 pVR1012x/s R5gp145 (dCFI)/h/dV14pVR1012x/s R5gp145 (dCFI) dv14/h 61 61 2867 pVR1012x/s R5gp145(dCFI)/h/dV23 pVR1012x/s R5gp145 (dCFI) dv23/h 62 62 2868 pVR1012x/sR5gp145 (dCFI)/h/dV24 pVR1012x/s R5gp145 (dCFI) dv24/h 63 63 2869pVR1012x/s R5gp145 (dCFI)/h/dV34 pVR1012x/s R5gp145 (dCFI) dv34/h 64 642870 pVR1012x/s R5gp145 (dCFI)/h/dV134 pVR1012x/s R5gp145 (dCFI) dv134/h65 65 2871 pVR1012x/s R5gp145 (dCFI)/h/dV234 pVR1012x/s R5gp145 (dCFI)dv234/h 66 66 2872 pVR1012x/s R5gp145 (dCFI) dv123/h pVR1012x/s R5gp145(dCFI) dv123/h 67 67 2873 pVR1012x/s R5gp145 (dCFI)/h/dV124 pVR1012x/sR5gp145 (dCFI) dv124/h 68 68 2874 pVR1012x/s R5gp145 (dCFI)/h/dV1234pVR1012x/s R5gp145 (dCFI) dv1234/h 69 69 2900 pVR1012x/s R5gp150/hpVR1012x/s R5gp150/h 70 70 3000 pVR1012x/s R5gp160/h pVR1012x/sR5gp160/h71 71 3200 pVR1012x/s X4gp150/h pVR1012x/s X4gp150/h 72 72 3201pVR1012x/s X4gp150 ΔCFI/h OR pVR1012x/s X4gp150 (del pVR1012x/s X4gp150(del F/CL del H IS)/h 73 73 F/CL del H IS)/h 3202 pVR1012x/s X4gp150Δgly/h pVR1012x/s X4gp150 delta gly 74 74 3203 pVR1012x/s X4gp150 ABΔgly/h pVR1012x/s X4gp150 AB (delta) gly/h 75 75 3300 pVR1012x/sX4gp160/h pVR1012x/s X4gp160/h 76 76 3301 pVR1012x/s X4gp160 ΔCFI/h ORpVR1012x/s X4gp160 (del pVR1012x/s X4gp160 (del F/CL del H IS)/h 77 77F/CL del H IS)/h 3400 pVR1012x/s X4gp160 Δgly/h pVR1012x/s X4gp160 deltagly 78 78 3401 pVR1012x/s X4gp160 AB Δgly/h OR pVR1012x/s pVR1012x/sX4gp160 AB Dgly/h 79 79 X4gp160AB mut Δgly/h 3500 pVR1012x/s Nef/hpVR1012x/s Nef/h 80 80 3600 pVR1012x/s NefDMHCDCD4/h pVR1012x/s Nefdelta MHC delta CD4/h 81 81 3700 pVR1012x/s NefDCD4/h pVR1012x/s Nefdelta CD4/h 82 82 3800 pVR1012x/s NefDMHC/h pVR1012x/s Nef delta MHC/h83 83 5200 pVR1012 x/s 89.6Pgp128 (del F/CL)/h pVR1012 x/s 89.6Pgp128(del F/CL)/h 84 84 5201 R5 Clade 89.6P gp140 ΔCFI/h pVR1012x/s89.6Pgp140 (del F/CL del H IS)/h 85 85 5202 pVR1012 x/s 89.6Pgp145 (delF/CL del H IS)/h pVR1012 x/s 89.6Pgp145 (del F/CL del H IS)/h 86 86 5203pVR1012 x/s 89.6Pgp160/h pVR1012 x/s 89.6Pgp160/h 87 87 5300 R5 Clade Cgp140 ΔCFI/h OR pVR1012x/s R5 pVR1012x/s CladeC (R5) gp140 (del F/CL delH IS)/h 88 88 (cladeC) gp140 (del F/CL del H IS)/h 5301 pVR1012x/s R5(cladeC) gp145 (del F/CL del H IS)/h pVR1012x/s CladeC (R5) gp145 (delF/CL del H IS)/h 89 89 5303 pVR1012x/s R5gp145 CladeC (Brazil) delCFI/hpVR1012x/s R5gp145 CladeC (Brazil) delCFI/h 90 90 5304 pVR1012x/s R5(clade A) gp140 (del F/CL del H IS)/h pVR1012x/s R5gp140CladeA (dCFI)/h91 91 5305 pVR1012x/s R5 (clade A) gp145 (del F/CL del H IS)/hpVR1012x/s R5gp145CladeA (dCFI)/h 92 92 5306 pVR1012x/s R5 (clade E)gp140 (del F/CL del H IS)/h pVR1012x/s R5gp140 Clade E (dCFI)/h 93 935307 pVR1012x/s R5 (clade E) gp145 (del F/CL del H IS)/h pVR1012x/s R5gp145 Clade E (dCFI)/h 94 94 5308 pVR1012x/s R5 (clade C South African)gp140 (del F/CL del H pVR1012x/s R5gp140 Clade C (SA) (dCFI)/h 95 95IS)/h 5309 pVR1012x/s R5 (clade C South African) gp145 (del F/CL del HpVR1012x/s R5gp145 CladeC (SA) (dCFI)/h 96 96 IS)/h 5350 pVRC1012 (x/s)-gp140 (dCFI) (Brazil C)/dV1 pVR1012x/s R5gp140CladeC (Brazil) dCFIdv1/h97 97 5351 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV12 pVR1012x/sR5gp140CladeC (Brazil) (dCFI) dv12/h 98 98 5352 pVRC1012 (x/s) -gp140(dCFI) (Brazil C)/dV123 pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv123/h99 99 5353 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV1234 pVR1012x/sR5gp140CladeC (Brazil) (dCFI) dv1234/h 100 100 5354 pVRC1012 (x/s)-gp140 (dCFI) (Brazil C)/dV124 pVR1012x/s R5gp140CladeC (Brazil) (dCFI)dv124/h 101 101 5355 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV13pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv13/h 102 102 5356 pVRC1012(x/s) -gp140 (dCFI) (Brazil C)/dV134 pVR1012x/s R5gp140CladeC (Brazil)(dCFI) dv134/h 103 103 5357 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV14pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv14/h 104 104 5358 pVRC1012(x/s) -gp140 (dCFI) (Brazil C)/dV2 pVR1012x/s R5gp140CladeC (Brazil)(dCFI) dv2/h 105 105 5359 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV23pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv23/h 106 106 5360 pVRC1012(x/s) -gp140 (dCFI) (Brazil C)/dV234 pVR1012x/s R5gp140CladeC (Brazil)(dCFI) dv234/h 107 107 5361 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV24pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv24/h 108 108 5362 pVRC1012(x/s) -gp140 (dCFI) (Brazil C)/dV3 pVR1012x/s R5gp140CladeC (Brazil)(dCFI) dv3/h 109 109 5363 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV34pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv34/h 110 110 5364 pVRC1012(x/s) -gp140 (dCFI) (Brazil C)/dV4 pVR1012x/s R5gp140CladeC (Brazil)(dCFI) dv4/h 111 111 5365 pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV1pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv1/h 112 112 5366 pVRC1012(x/s) -gp145 (dCFI) (Brazil C)/dV12 pVR1012x/s R5gp145CladeC (Brazil)(dCFI) dv12/h 113 113 5367 pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV123pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv123/h 114 114 5368 pVRC1012(x/s) -gp145 (dCFI) (Brazil C)/dV1234 pVR1012x/s R5gp145CladeC (Brazil)(dCFI) dv1234/h 115 115 5369 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV124 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv124/h 116 116 5370pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV13 pVR1012x/s R5gp145CladeC(Brazil) (dCFI) dv13/h 117 117 5371 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV134 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv134/h 118 118 5372pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV14 pVR1012x/s R5gp145CladeC(Brazil) (dCFI) dv14/h 119 119 5373 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV2 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv2/h 120 120 5374pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV23 pVR1012x/s R5gp145CladeC(Brazil) (dCFI) dv23/h 121 121 5375 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV234 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv234/h 122 122 5376pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV24 pVR1012x/s R5gp145CladeC(Brazil) (dCFI) dv24/h 123 123 5377 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV3 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv3/h 124 124 5378pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV34 pVR1012x/s R5gp145CladeC(Brazil) (dCFI) dv34/h 125 125 5379 pVRC1012 (x/s) -gp145 (dCFI) (BrazilC)/dV4 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv4/h 126 126 5500pVR1012x/s R5 (SA-C) gp140 (dCFI) dV1/h pVR1012x/s R5 gp140 (dCFI) SAdv1/h 127 127 5501 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV12/h pVR1012x/sR5 gp140 (dCFI) SA dv12/h 128 128 5502 pVR1012x/s R5 (SA-C) gp140 (dCFI)dV123/h pVR1012x/s R5 gp140 (dCFI) SA dv123/h 129 129 5503 pVR1012x/s R5(SA-C) gp140 (dCFI) dV1234/h pVR1012x/s R5 gp140 (dCFI) SA dv1234/h 130130 5504 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV124/h pVR1012x/s R5 gp140(dCFI) SA dv124/h 131 131 5505 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV13/hpVR1012x/s R5 gp140 (dCFI) SA dv13/h 132 132 5506 pVR1012x/s R5 (SA-C)gp140 (dCFI) dV134/h pVR1012x/s R5 gp140 (dCFI) SA dv134/h 133 133 5507pVR1012x/s R5 (SA-C) gp140 (dCFI) dV14/h pVR1012x/s R5 gp140 (dCFI) SAdv14/h 134 134 5508 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV2/h pVR1012x/sR5 gp140 (dCFI) SA dv2/h 135 135 5509 pVR1012x/s R5 (SA-C) gp140 (dCFI)dV23/h pVR1012x/s R5 gp140 (dCFI) SA dv23/h 136 136 5510 pVR1012x/s R5(SA-C) gp140 (dCFI) dV234/h pVR1012x/s R5 gp140 (dCFI) SA dv234/h 137137 5511 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV24/h pVR1012x/s R5 gp140(dCFI) SA dv24/h 138 138 5512 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV3/hpVR1012x/s R5 gp140 (dCFI) SA dv3/h 139 139 5513 pVR1012x/s R5 (SA-C)gp140 (dCFI) dV34/h pVR1012x/s R5 gp140 (dCFI) SA dv34/h 140 140 5514pVR1012x/s R5 (SA-C) gp140 (dCFI) dV4/h pVR1012x/s R5 gp140 (dCFI) SAdv4/h 141 141 5515 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV1/h pVR1012x/s R5gp145 (dCFI) SA dv1/h 142 142 5516 pVR1012x/s R5 (SA-C) gp145 (dCFI)dV12/h pVR1012x/s R5 gp145 (dCFI) SA dv12/h 143 143 5517 pVR1012x/s R5(SA-C) gp145 (dCFI) dV123/h pVR1012x/s R5 gp145 (dCFI) SA dv123/h 144144 5518 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV1234/h pVR1012x/s R5 gp145(dCFI) SA dv1234/h 145 145 5519 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV2/hpVR1012x/s R5 gp145 (dCFI) SA dv2/h 146 146 5520 pVR1012x/s R5 (SA-C)gp145 (dCFI) dV23/h pVR1012x/s R5 gp145 (dCFI) SA dv23/h 147 147 5521pVR1012x/s R5 (SA-C) gp145 (dCFI) dV234/h pVR1012x/s R5 gp145 (dCFI) SAdv234/h 148 148 5522 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV24/h pVR1012x/sR5 gp145 (dCFI) SA dv24/h 149 149 5523 pVR1012x/s R5 (SA-C) gp145 (dCFI)dV3/h pVR1012x/s R5 gp145 (dCFI) SA dv3/h 150 150 5524 pVR1012x/s R5(SA-C) gp145 (dCFI) dV34/h pVR1012x/s R5 gp145 (dCFI) SA dv34/h 151 1515525 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV4/h pVR1012x/s R5 gp145 (dCFI)SA dv4/h 152 152 5526 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV13/hpVR1012x/s R5 gp145 (dCFI) SA dv13/h 153 153 5527 pVR1012x/s R5 (SA-C)gp145 (dCFI) dV134/h pVR1012x/s R5 gp145 (dCFI) SA dv134/h 154 154 5528pVR1012x/s R5 (SA-C) gp145 (dCFI) dV124/h pVR1012x/s R5 gp145 (dCFI) SAdv124/h 155 155 5529 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV14/h pVR1012x/sR5 gp145 (dCFI) SA dv14/h 156 156 Gag/Pol plasmids 3900 pVR1012x/s HIVGag/h pVR1012x/s Gag/h 157 157 3901 pVR1012x/s SIV Gag/h pVR1012x/s SIVGag/h 158 158 4000 pVR1012x/s HIV Gag-Pol ΔFS ΔPR/h OR pVR1012x/s Gag-pVR1012x/s Gag-PoldeltaFSdeltaPr/h 159 159 Pol/h 4001 pVR1012x/s SIVGag-Pol/h pVR1012x/s SIV Gag-Pol/h 160 160 4100 pVR1012x/s HIV Pol/hpVR1012x/s Pol/h 161 161 4101 pVR1012x/s SIV Pol/h pVR1012x/s SIV Pol/h162 162 4200 pVR1012x/s HIV Gag-Pol/h pVR1012x/s Gag (fs) Pol/h 163 1634300 pVR1012x/s HIV Gag-Pol ΔRT ΔIN/h OR pVR1012 Gag- pVR1012x/s Gag-Pol(fs) RT (−) IN (−) 164 164 Pol (d delta RT delta IN)/h 4301pVR1012x/s-Gag (FS) -Pol-delta RT IN-IRES-R5 gp157-Nef pVR1012x/s-Gag(FS) -Pol-delta RT IN-IRES-R5 gp157-Nef 165 165 4302 pVR1012x/s HIVgag-Pol ΔFS ΔPR ΔRT ΔIN/h pVR1012x/s Gag (delFS) Pol (delta PR delta RTdelta IN)/h 166 166 4303 pVR1012x/s SIV Gag-Pol ΔFS/h OR pVR1012 SIVpVR1012x/s SIV Gag (delFS) -Pol/h 167 167 Gag (delFS) Pol (delta PRdelta RT delta IN)/h 4304 pVR1012 Gag (delFS) Pol delta PR delta RTdelta IN/h pVR1012 Gag-C (delFS) Pol (deltaPR deltaRT deltaIN)/h 168 1684305 pVR1012 Gag-A (delFS) Pol (delta PR delta RT delta IN)/h pVR1012Gag-A (delFS) Pol (deltaPR delta RT deltaIN)/h 169 169 4306 pVR1012 Gag(delFS) Pol delta PR delta RT delta IN/Nef/h pVR1012 Gag (delFS) PoldeltaPR deltaRT deltaIN/Nef/h 170 170 4308 pVR1012 Gag (delFS) PoldeltaPR deltaRT deltaIN deltaMyr/h pVR1012 Gag (delFS) Pol deltaPRdeltaRT deltaIN delta 171 171 Myr/h 4309 pVR1012 Gag (delFS) Pol deltaPRdeltaRT deltaIN delta pVR1012x/s Gag (delFS) Pol deltaPR deltaRT deltaINdelta 172 172 Myr/Nef/h Myr/Nef/h 4310 pVR1012 Nef Gag (del fs) (delMyr) Pol (delta PR delta RT pVR1012 Nef Gag (delFS) (del Myr) Pol (deltaPR delta RT 173 173 delta IN)/h delta IN)/h 4311 pVR1012 Gag-C (delFS)Pol (delta PR delta RT delta IN) Nef/h pVR1012 Gag-C (del PS) Pol (deltaPR dalta RT delta IN) 174 174 Nef/h 4312 Gag (del fs) (del Myr) Nef PolΔPRΔRTΔIN/h pVR1012 Gag (del FS) (del Myr) Nef Pol (delta PR delta RT175 175 delta IN)/h 4313 pVR1012 Gag Clade A (del fs) Pol (Δ PR Δ RT ΔIN)/h pVR1012 Gag-A (del FS) Pol (delPR delRT delIN) Nef/h 176 176

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides modifications of HIV Env, Gag, Pol, andNef that enhance immunogenicity for genetic immunization. Both HIV andSIV are genetically related members of the lentivirus genus of theRetroviridae family. Lentivirus isolates from humans are grouped intoone of two types, designated HIV-1 and HIV-2. A classification schemerecognizes nine subtypes (clades) of HIV-1 (A through 1) and fivesubtypes of HIV-2 (A through E). A compendium of HIV and SIV sequenceinformation is found in a database prepared by Myers et al., Los Alamos,N. Mex.: Los Alamos National Laboratory.

Nucleic Acid Molecules

As indicated herein, nucleic acid molecules of the present invention maybe in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA may be double-stranded orsingle-stranded. Single-stranded DNA or RNA may be the coding strand,also known as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Nucleic acid molecules of the present invention include DNA moleculescomprising an open reading frame (ORF) of a wild-type HIV gene; and DNAmolecules which comprise a sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode an ORF of a wild-type HIV polypeptide. Of course, thegenetic code is well known in the art. Degenerate variants optimized forhuman codon usage are preferred.

In another aspect, the invention provides a nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above. By “stringenthybridization conditions” is intended overnight incubation at 42 degreeC. in a solution comprising: 50% formamide, 5 times SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 timesDenhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1 timesSSC at about 65 degree C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide. Of course, apolynucleotide which hybridizes only to a complementary stretch of T (orU) resides, would not be included in a polynucleotide of the inventionused to hybridize to a portion of a nucleic acid of the invention, sincesuch a polynucleotide would hybridize to any nucleic acid moleculecontaining a poly T (or U) stretch or the complement thereof (e.g.,practically any double-stranded DNA clone).

As indicated herein, nucleic acid molecules of the present inventionwhich encode an HIV polypeptide may include, but are not limited tothose encoding the amino acid sequence of the full-length polypeptide,by itself, the coding sequence for the full-length polypeptide andadditional sequences, such as those encoding a leader or secretorysequence, such as a pre-, or pro- or prepro-protein sequence, the codingsequence of the full-length polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, for example,ribosome binding and stability of mRNA; and additional coding sequencewhich codes for additional amino acids, such as those which provideadditional functionalities.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the HIV protein. Variants may occur naturally, such as anatural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on a genome ofan organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York(1985). Non-naturally occurring variants may be produced using art-knownmutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the HIVpolypeptide or portions thereof. Also especially preferred in thisregard are conservative substitutions.

Further embodiments of the invention include nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 95%identical, and more preferably at least 96%, 97%, 98% or 99% identicalto a nucleotide sequence encoding a polypeptide having the amino acidsequence of a wild-type HIV polypeptide or a nucleotide sequencecomplementary thereto.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a HIVpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the HIVpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to the reference nucleotidesequence can be determined conventionally using known computer programssuch as the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between twosequences. When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, of course, such that the percentage of identity iscalculated over the full length of the reference nucleotide sequence andthat gaps in homology of up to 5% of the total number of nucleotides inthe reference sequence are allowed.

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shownherein in the Sequence Listing which encode a polypeptide having HIVpolypeptide activity. By “a polypeptide having HIV activity” is intendedpolypeptides exhibiting HIV activity in a particular biological assay.For example, Env, Gag, and Pol protein activity can be measured forchanges in immunological character by an appropriate immunologicalassay.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or99% identical to a nucleic acid sequence shown herein in the SequenceListing will encode a polypeptide “having HIV polypeptide activity.” Infact, since degenerate variants of these nucleotide sequences all encodethe same polypeptide, this will be clear to the skilled artisan evenwithout performing the above described comparison assay. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having HIV polypeptide activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Polypeptides and Fragments

The invention further provides a HIV polypeptide having the amino acidsequence encoded by an open reading frame (ORF) of a wild-type HIV gene,or a peptide or polypeptide comprising a portion thereof (e.g., gp120).

It will be recognized in the art that some amino acid sequences of theHIV polypeptides can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

Thus, the invention further includes variations of the HIV polypeptidewhich show substantial HIV polypeptide activity or which include regionsof HIV protein such as the protein portions discussed below. Suchmutants include deletions, insertions, inversions, repeats, and typesubstitutions. As indicated, guidance concerning which amino acidchanges are likely to be phenotypically silent can be found in Bowie, J.U., et al., “Deciphering the Message in Protein Sequences: Tolerance toAmino Acid Substitutions,” Science 247:1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptide of theinvention may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which additional amino acids arefused to the mature polypeptide, such as an IgG Fc fusion region peptideor leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table A).

TABLE A Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Ionizable: Acidic Aspartic Acid Glutamic AcidIonizable: Basic Arginine Histidine Lysine Nonionizable Polar AsparagineGlutamine Selenocystine Serine Threonine Nonpolar (Hydrophobic) AlanineGlycine Isoleucine Leucine Proline Valine Sulfur Containing CysteineMethionine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions for any givenHIV polypeptide will not be more than 50, 40, 30, 20, 10, 5 or 3.

Amino acids in the HIV polypeptides of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as changes in immunological character.

The polypeptides of the present invention are conveniently provided inan isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention.

Also intended as an “isolated polypeptide” are polypeptides that havebeen purified, partially or substantially, from a recombinant host cellor a native source. For example, a recombinantly produced version of theHIV polypeptide can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include a polypeptidecomprising a polypeptide shown herein in the Sequence Listing; as wellas polypeptides which are at least 95% identical, and more preferably atleast 96%, 97%, 98% or 99% identical to those described above and alsoinclude portions of such polypeptides with at least 30 amino acids andmore preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an HIV polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the HIV polypeptide. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acidsequence shown herein in the Sequence Listing can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

The polypeptides of the invention may be produced by any conventionalmeans. Houghten, R. A. (1985) General method for the rapid solid-phasesynthesis of large numbers of peptides: specificity of antigen-antibodyinteraction at the level of individual amino acids. Proc. Natl. Acad.Sci. USA 82:5131-5135. This “Simultaneous Multiple Peptide Synthesis(SMPS)” process is further described in U.S. Pat. No. 4,631,211 toHoughten et al. (1986).

The present invention also relates to vectors which include the nucleicacid molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof HIV polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The HIV polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include naturally purified products, products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

Pharmaceutical Formulations, Dosages, and Modes of Administration

The compounds of the invention may be administered using techniques wellknown to those in the art. Preferably, compounds are formulated andadministered by genetic immunization. Techniques for formulation andadministration may be found in “Remington's Pharmaceutical Sciences”,18^(th) ed., 1990, Mack Publishing Co., Easton, Pa. Suitable routes mayinclude parenteral delivery, such as intramuscular, intradermal,subcutaneous, intramedullary injections, as well as, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few. For injection, the compoundsof the invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer.

In instances wherein intracellular administration of the compounds ofthe invention is preferred, techniques well known to those of ordinaryskill in the art may be utilized. For example, such compounds may beencapsulated into liposomes, then administered as described above.Liposomes are spherical lipid bilayers with aqueous interiors. Allmolecules present in an aqueous solution at the time of liposomeformation are incorporated into the aqueous interior. The liposomalcontents are both protected from the external microenvironment and,because liposomes fuse with cell membranes, are effectively deliveredinto the cell cytoplasm.

Nucleotide sequences of the invention which are to be intracellularlyadministered may be expressed in cells of interest, using techniqueswell known to those of skill in the art. For example, expression vectorsderived from viruses such as retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, or sindbis or other RNA viruses, or from plasmids may be usedfor delivery and expression of such nucleotide sequences into thetargeted cell population. Methods for the construction of suchexpression vectors are well known. See, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., and Ausubel et al., 1989, Current Protocols InMolecular Biology, Greene Publishing Associates and Wiley Interscience,NY.

The invention extends to the use of a plasmid for primary immunization(priming) of a host and the subsequent use of a recombinant virus, suchas a retrovirus, adenovirus, adeno-associated virus, herpes virus,vaccinia virus, polio virus, or sindbis or other RNA virus, for boostingsaid host, and vice versa. For example, the host may be immunized(primed) with a plasmid by DNA immunization and receive a boost with thecorresponding viral construct, and vice versa. Alternatively, the hostmay be immunized (primed) with a plasmid by DNA immunization and receivea boost with not the corresponding viral construct but a different viralconstruct, and vice versa.

With respect to HIV Env, Gag, and Pol, protein sequences of theinvention may be used as therapeutics or prophylatics (as subunitvaccines) in the treatment of AIDS or HIV infection. A therapeuticallyeffective dose refers to that amount of the compound sufficient toresult in amelioration of symptoms or a prolongation of survival in apatient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that includes the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (e.g., the concentration ofthe test compound which achieves a half-maximal inhibition of viralinfection relative to the amount of the event in the absence of the testcompound) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography(HPLC).

The compounds of the invention may, further, serve the role of aprophylactic vaccine, wherein the host produces antibodies and/or CTLresponses against HIV Env, Gag and Pol, which responses then preferablyserve to neutralize HIV viruses by, for example, inhibiting further HIVinfection. Administration of the compounds of the invention as aprophylactic vaccine, therefore, would comprise administering to a hosta concentration of compounds effective in raising an immune responsewhich is sufficient to elicit antibody and/or CTL responses to HIV Env,Gag, and Pol, and/or neutralize HIV, by, for example, inhibiting HIVability to infect cells. The exact concentration will depend upon thespecific compound to be administered, but may be determined by usingstandard techniques for assaying the development of an immune responsewhich are well known to those of ordinary skill in the art.

The compounds may be formulated with a suitable adjuvant in order toenhance the immunological response. Such adjuvants may include, but arenot limited to mineral gels such as aluminum hydroxide; surface activesubstances such as lysolecithin, pluronic polyols, polyanions; otherpeptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

Adjuvants suitable for co-administration in accordance with the presentinvention should be ones that are potentially safe, well tolerated andeffective in people including QS-21, Detox-PC, MPL-SE, MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59(see Kim et al., 2000, Vaccine, 18: 597 and references therein).

Other contemplated adjuvants that may be administered include lectins,growth factors, cytokines and lymphokines such as alpha-interferon,gamma-interferon, platelet derived growth factor (PDGF), gCSF, gMCSF,TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10and IL-12.

For all such treatments described above, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administereddose in the management of the viral infection of interest will vary withthe severity of the condition to be treated and the route ofadministration. The dose and perhaps prime-boost regimen, will also varyaccording to the age, weight, and response of the individual patient. Aprogram comparable to that discussed above may be used in veterinarymedicine.

The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of galenic pharmacy toproduce medicinal agents for administration to patients, e.g., mammalsincluding humans.

The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application which do not deleteriously react with theactive compounds. Suitable pharmaceutically acceptable carriers includebut are not limited to water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatine,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., vitamins.

For parenteral application, which includes intramuscular, intradermal,subcutaneous, intranasal, intracapsular, intraspinal, intrasternal, andintravenous injection, particularly suitable are injectable, sterilesolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants, including suppositories. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules. The pharmaceuticalcompositions may be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate. A syrup, elixir, or the like can beused wherein a sweetened vehicle is employed.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active compound is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze dry the newcompounds and use the lyophilizates obtained, for example, for thepreparation of products for injection.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For topical application, there are employed as non-sprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, aerosols, etc., which are, if desired, sterilized ormixed with auxiliary agents, e.g., preservatives, stabilizers, wettingagents, buffers or salts for influencing osmotic pressure, etc. Fortopical application, also suitable are sprayable aerosol preparationswherein the active ingredient, preferably in combination with a solid orliquid inert carrier material, is packaged in a squeeze bottle or inadmixture with a pressurized volatile, normally gaseous propellant,e.g.; a freon.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Genetic Immunization

Genetic immunization according to the present invention elicits aneffective immune response without the use of infective agents orinfective vectors. Vaccination techniques which usually do produce a CTLresponse do so through the use of an infective agent. A complete, broadbased immune response is not generally exhibited in individualsimmunized with killed, inactivated or subunit vaccines. The presentinvention achieves the full complement of immune responses in a safemanner without the risks and problems associated with vaccinations thatuse infectious agents.

According to the present invention, DNA or RNA that encodes a targetprotein is introduced into the cells of an individual where it isexpressed, thus producing the target protein. The DNA or RNA is linkedto regulatory elements necessary for expression in the cells of theindividual. Regulatory elements for DNA include a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct.

The genetic constructs of genetic vaccines comprise a nucleotidesequence that encodes a target protein operably linked to regulatoryelements needed for gene expression. Accordingly, incorporation of theDNA or RNA molecule into a living cell results in the expression of theDNA or RNA encoding the target protein and thus, production of thetarget protein.

When taken up by a cell, the genetic construct which includes thenucleotide sequence encoding the target protein operably linked to theregulatory elements may remain present in the cell as a functioningextrachromosomal molecule or it may integrate into the cell'schromosomal DNA. DNA may be introduced into cells where it remains asseparate genetic material in the form of a plasmid. Alternatively,linear DNA which can integrate into the chromosome may be introducedinto the cell. When introducing DNA into the cell, reagents whichpromote DNA integration into chromosomes may be added. DNA sequenceswhich are useful to promote integration may also be included in the DNAmolecule. Since integration into the chromosomal DNA necessarilyrequires manipulation of the chromosome, it is preferred to maintain theDNA construct as a replicating or non-replicating extrachromosomalmolecule. This reduces the risk of damaging the cell by splicing intothe chromosome without affecting the effectiveness of the vaccine.Alternatively, RNA may be administered to the cell. It is alsocontemplated to provide the genetic construct as a linear minichromosomeincluding a centromere, telomeres and an origin of replication.

The necessary elements of a genetic construct of a genetic vaccineinclude a nucleotide sequence that encodes a target protein and theregulatory elements necessary for expression of that sequence in thecells of the vaccinated individual. The regulatory elements are operablylinked to the DNA sequence that encodes the target protein to enableexpression.

The molecule that encodes a target protein is a protein-encodingmolecule which is translated into protein. Such molecules include DNA orRNA which comprise a nucleotide sequence that encodes the targetprotein. These molecules may be cDNA, genomic DNA, synthesized DNA or ahybrid thereof or an RNA molecule such as mRNA. Accordingly, as usedherein, the terms “DNA construct”, “genetic construct” and “nucleotidesequence” are meant to refer to both DNA and RNA molecules.

The regulatory elements necessary for gene expression of a DNA moleculeinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression. It is necessary that these elements be operable in thevaccinated individual. Moreover, it is necessary that these elements beoperably linked to the nucleotide sequence that encodes the targetprotein such that the nucleotide sequence can be expressed in the cellsof a vaccinated individual and thus the target protein can be produced.

Initiation codons and stop codons are generally considered to be part ofa nucleotide sequence that encodes the target protein. However, it isnecessary that these elements are functional in the vaccinatedindividual.

Similarly, promoters and polyadenylation signals used must be functionalwithin the cells of the vaccinated individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, can be used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pCEP4 and pREP4from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. When theconstruct is introduced into the cell, tk will be produced. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk. Thus, a system canbe provided which allows for the selective destruction of vaccinatedcells.

In order to be a functional genetic construct, the regulatory elementsmust be operably linked to the nucleotide sequence that encodes thetarget protein. Accordingly, it is necessary for the initiation andtermination codons to be in frame with the coding sequence.

Open reading frames (ORFs) encoding the protein of interest and anotheror other proteins of interest may be introduced into the cell on thesame vector or on different vectors. ORFs on a vector may be controlledby separate promoters or by a single promoter. In the latterarrangement, which gives rise to a polycistronic message, the ORFs willbe separated by translational stop and start signals. The presence of aninternal ribosome entry site (IRES) site between these ORFs permits theproduction of the expression product originating from the second ORF ofinterest, or third, etc. by internal initiation of the translation ofthe bicistronic or polycistronic mRNA.

According to the invention, the genetic vaccine may be administereddirectly into the individual to be immunized or ex vivo into removedcells of the individual which are reimplanted after administration. Byeither route, the genetic material is introduced into cells which arepresent in the body of the individual. Routes of administration include,but are not limited to, intramuscular, intraperitoneal, intradermal,subcutaneous, intravenous, intraarterially, intraoccularly and oral aswell as transdermally or by inhalation or suppository. Preferred routesof administration include intramuscular, intraperitoneal, intradermaland subcutaneous injection. Genetic constructs may be administered bymeans including, but not limited to, traditional syringes, needlelessinjection devices, or microprojectile bombardment gene guns.Alternatively, the genetic vaccine may be introduced by various meansinto cells that are removed from the individual. Such means include, forexample, ex vivo transfection, electroporation, microinjection andmicroprojectile bombardment. After the genetic construct is taken up bythe cells, they are reimplanted into the individual. It is contemplatedthat otherwise non-immunogenic cells that have genetic constructsincorporated therein can be implanted into the individual even if thevaccinated cells were originally taken from another individual.

The genetic vaccines according to the present invention comprise about 1nanogram to about 1000 micrograms of DNA. In some preferred embodiments,the vaccines contain about 10 nanograms to about 800 micrograms of DNA.In some preferred embodiments, the vaccines contain about 0.1 to about500 micrograms of DNA. In some preferred embodiments, the vaccinescontain about 1 to about 350 micrograms of DNA. In some preferredembodiments, the vaccines contain about 25 to about 250 micrograms ofDNA. In some preferred embodiments, the vaccines contain about 100micrograms DNA.

The genetic vaccines according to the present invention are formulatedaccording to the mode of administration to be used. One having ordinaryskill in the art can readily formulate a genetic vaccine that comprisesa genetic construct. In cases where intramuscular injection is thechosen mode of administration, an isotonic formulation is preferablyused. Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are preferred. Stabilizersinclude gelatin and albumin. In some embodiments, a vaso-constrictionagent is added to the formulation. The pharmaceutical preparationsaccording to the present invention are provided sterile and pyrogenfree.

Genetic constructs may optionally be formulated with one or moreresponse enhancing agents such as: compounds which enhance transfection,i.e. transfecting agents; compounds which stimulate cell division, i.e.replication agents; compounds which stimulate immune cell migration tothe site of administration, i.e. inflammatory agents; compounds whichenhance an immune response, i.e. adjuvants or compounds having two ormore of these activities.

In one embodiment, bupivacaine, a well known and commercially availablepharmaceutical compound, is administered prior to, simultaneously withor subsequent to the genetic construct. Bupivacaine and the geneticconstruct may be formulated in the same composition. Bupivacaine isparticularly useful as a cell stimulating agent in view of its manyproperties and activities when administered to tissue. Bupivacainepromotes and facilitates the uptake of genetic material by the cell. Assuch, it is a transfecting agent. Administration of genetic constructsin conjunction with bupivacaine facilitates entry of the geneticconstructs into cells. Bupivacaine is believed to disrupt or otherwiserender the cell membrane more permeable. Cell division and replicationis stimulated by bupivacaine. Accordingly, bupivacaine acts as areplicating agent. Administration of bupivacaine also irritates anddamages the tissue. As such, it acts as an inflammatory agent whichelicits migration and chemotaxis of immune cells to the site ofadministration. In addition to the cells normally present at the site ofadministration, the cells of the immune system which migrate to the sitein response to the inflammatory agent can come into contact with theadministered genetic material and the bupivacaine. Bupivacaine, actingas a transfection agent, is available to promote uptake of geneticmaterial by such cells of the immune system as well.

In addition to bupivacaine, mepivacaine, lidocaine, procains,carbocaine, methyl bupivacaine, and other similarly acting compounds maybe used as response enhancing agents. Such agents acts a cellstimulating agents which promote the uptake of genetic constructs intothe cell and stimulate cell replication as well as initiate aninflammatory response at the site of administration.

Other contemplated response enhancing agents which may function astransfecting agents and/or replicating agents and/or inflammatory agentsand which may be administered include lectins, growth factors, cytokinesand lymphokines such as alpha-interferon, gamma-interferon, plateletderived growth factor (PDGF), gCSF, gMCSF, TNF, epidermal growth factor(EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12 as well ascollagenase, fibroblast growth factor, estrogen, dexamethasone,saponins, surface active agents such as immune-stimulating complexes(ISCOMS), Freund's incomplete adjuvant, LPS analog includingmonophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs andvesicles such as squalene and squalane, hyaluronic acid andhyaluronidase may also be used administered in conjunction with thegenetic construct. In some embodiments, combinations of these agents areco-administered in conjunction with the genetic construct. In otherembodiments, genes encoding these agents are included in the same ordifferent genetic construct(s) for co-expression of the agents.

With respect to HIV Env, Gag, and Pol nucleotide sequences of theinvention, particularly through genetic immunization, may be used astherapeutics or prophylatics in the treatment of AIDS or HIV infection.A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies can be used in formulating a range of dosagefor use in humans. The dosage of such compounds lies preferably within arange of circulating concentrations that includes the ED50 with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the (e.g., the concentration of thetest compound which achieves a half-maximal inhibition of viralinfection relative to the amount of the event in the absence of the testcompound) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography(HPLC).

The compounds (for genetic immunization) of the invention may, further,serve the role of a prophylactic vaccine, wherein the host producesantibodies and/or CTL responses against HIV Env, Gag, and Pol whichresponses then preferably serve to neutralize HIV viruses by, forexample, inhibiting further HIV infection. Administration of thecompounds of the invention as a prophylactic vaccine, therefore, wouldcomprise administering to a host a concentration of compounds effectivein raising an immune response which is sufficient to elicit antibodyand/or CTL responses to HIV Env, Gag, and Pol and/or neutralize HIV, by,for example, inhibiting HIV ability to infect cells. The exactconcentration will depend upon the specific compound to be administered,but may be determined by using standard techniques for assaying thedevelopment of an immune response which are well known to those ofordinary skill in the art.

Env

To improve the immune response to native gp160 and to expose the coreprotein for optimal antigen presentation and recognition, we haveanalyzed the immune response to modified forms of the protein. The roleof conserved N-linked glycosylation sites has been studied, andanalogues of fusion intermediates have been developed. Expressionvectors with deletions in the cleavage site (C), the fusion peptide (F),and the interspace (I) between the two heptad repeats were termed ΔCFI.Plasmid DNA vaccination has been a useful technology for the developmentand analysis of immunogens. This method of vaccination allowsappropriate post-translational modification, proper intracellulartrafficking, and antigen presentation. Direct injection of naked DNAeither intramuscularly or intradermally in rodents induces immuneresponses, and the ability to easily modify plasmid expression vectorsto express different forms of HIV envelope proteins enables rapid andsystematic testing of vaccine immunogens. In this disclosure, we haveanalyzed the immune response to modified Env candidates expressed inplasmids with modified codons to improve gene expression. Both antibodyand CTL responses were analyzed after injection of plasmid DNA intomuscle. A modified gp140 DNA with improved ability to elicit antibodyand CTL responses to HIV Env has now been identified that is envisionedas a prototype immunogen that can elicit broadly neutralizing antibodyresponses to HIV.

Exposing the Core Protein of Viral Membrane Fusion Proteins

Described herein are modified HIV envelope proteins that improve theimmune response to native gp160 and expose the core protein for optimalantigen presentation and recognition. Weissenhorn et al., MolecularCell, 2, 605-616, 1998 proposes a core protein as a model for a fusionintermediate of viral glycoproteins, where the glycoproteins arecharacterized by a central triple stranded coiled coil followed by adisulfide-bonded loop that reverses the chain direction and connects toan α helix packed antiparallel to the core helices, as, for example, inthe case of Ebola Zaire GP2, Murine Moloney Leukemia virus (MuMoLv)55-residue segment of the TM subunit (Mo-55), low-pH-treated influenzaHA2, protease resistant core of HIV gp41, and SIV gp41 (FIG. 177). Thus,the strategy for improving the immune response by exposing the proteaseresistant core of HIV gp41 extends to other viral membrane fusionproteins that are characterized by a central triple stranded coiled coilfollowed by a disulfide-bonded loop that reverses the chain directionand connects to an ax helix packed antiparallel to the core helices.

The present approach involves a series of internal mutations designed toreplace the cleavage site (C), the fusion domain (F), and the interspace(I) between the two heptad repeats all on a backbone of COOH-terminaltruncations to expose the core protein of the viral membrane fusionprotein Env, based on modified gp140 as a prototype immunogen. Byreplacement is meant deletions, insertions, and/or substitutions ofamino acid residues. In one embodiment, deletions are meant (i.e., aminoacids are deleted to create the ΔCFI mutations).

In this embodiment, the ΔC mutation is intended to eliminate proteolysisby deleting the gp120/gp41 cleavage site that links the envelopecovalently to the ectodomain by 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81,%, 80%, 79%,78%, 77%, 76%, 75%, 74%, 73,%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%,64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%,50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42, 41%, 40%, 39%, 38%, 37%,36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1%.

In this embodiment, the ΔF mutation is intended to solubilize themolecule by deleting the fusion domain by 100%, 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81,%,80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%, 72%, 71%, 70%, 69%, 68%, 67%,66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%,52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42, 41%, 40%, 39%,38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%,24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

In this embodiment, the ΔI mutation is intended to stabilize oligomerformation by deleting the interspace between the two heptad repeats by100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%,72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%,58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%,44%, 43%, 42, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or1%.

In this embodiment, the COOH-terminal truncation is intended to reducetoxicity by deleting the cytoplasmic domain by 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%, 72%, 71%, 70%, 69%, 68%,67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%,53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42, 41%, 40%,39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%,25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

In this embodiment, optionally, the COOH-terminal truncation is extendedso as to solubilize the molecule by deleting the transmembrane domain by100% 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%,72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%,58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%,44%, 43%, 42, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or1%.

Amino acid substitutions may encompass those of a conserved ornon-conserved nature. Presumably, a non-conserved substitution of adomain would act like a deletion of the domain. Conserved amino acidsubstitutions constitute switching one or more amino acids with aminoacids of similar charge, size, and/or hydrophobicity characteristics.Non-conserved amino acid substitutions constitute switching one or moreamino acids with amino acids of dissimilar charge, size, and/orhydrophobicity characteristics. The families of amino acids include thebasic amino acids (lysine, arginine, histidine); the acidic amino acids(aspartic acid, glutamic acid); the non-polar amino acids (alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan); the uncharged polar amino acids (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine); and the aromaticamino acids (phenylalanine, tryptophan, and tyrosine). One or moresubstitutions may be introduced to achieve the ΔC mutation intended toeliminate proteolysis by acting like a deletion of the gp120/gp41cleavage site to link the envelope covalently to the ectodomain, the ΔFmutation intended to solubilize the molecule by acting like a deletionof the fusion domain, the ΔI mutation intended to stabilize oligomerformation by acting like a deletion of the interspace between the twoheptad repeats, the COOH-terminal truncation intended to reduce toxicityby acting like a deletion of the cytoplasmic domain, and, optionally,the COOH-terminal truncation extended so as to solubilize the moleculeby acting like a deletion of the transmembrane domain.

Amino acid insertions may constitute single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of a domain, as well as at a position internal to thedomain. Such insertions will generally range from 2 to 15 amino acids inlength. One or more insertions may be introduced to achieve the ΔCmutation intended to eliminate proteolysis by acting like a deletion ofthe gp120/gp41 cleavage site to link the envelope covalently to theectodomain, the AF mutation intended to solubilize the molecule byacting like a deletion of the fusion domain, the ΔI mutation intended tostabilize oligomer formation by acting like a deletion the interspacebetween the two heptad repeats, the COOH-terminal truncation intended toreduce toxicity by acting like a deletion of the cytoplasmic domain,and, optionally, the COOH-terminal truncation extended so as tosolubilize the molecule by acting like a deletion of the transmembranedomain.

The nucleic acids of the present invention are optionally DNA, RNA, ormRNA. Most typically, the nucleic acids are provided by recombinantlymaking a DNA, which is expressed in a cell as RNA and/or as mRNA. Giventhe strategy for making the nucleic acids of the present invention, oneof skill can construct a variety of clones containing functionallyequivalent nucleic acids. Cloning methodologies to accomplish theseends, and sequencing methods to verify the sequence of nucleic acids arewell known in the art. Examples of appropriate cloning and sequencingtechniques, and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, volume 152,Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Productinformation from manufacturers of biological reagents and experimentalequipment also provide information useful in known biological methods.Such manufacturers include the SIGMA chemical company (Saint Louis,Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill.

The nucleic acid compositions of this invention, whether RNA, cDNA,mRNA, genomic DNA, or a hybrid of the various combinations, are isolatedfrom biological sources or synthesized in vitro. The nucleic acids ofthe present invention are present in transformed or transfected wholecells, in transformed or transfected cell lysates, or in a partiallypurified or substantially pure form.

In vitro amplification techniques suitable for amplifying sequences toprovide a nucleic acid or for subsequent analysis, sequencing orsubcloning are known. Examples of techniques sufficient to directpersons of skill through such in vitro amplification methods, includingthe polymerase chain reaction (PCR) the ligase chain reaction (LCR),Qβ-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well asMullis, et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis, et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh, et al., Proc.Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli, et al., Proc. Natl.Acad. Sci. USA, 87:1874 (1990); Lomell, et al., J. Clin. Chem., 35:1826(1989); Landegren, et al., Science, 241:1077-1080 (1988); Van Brunt,Biotechnology, 8:291-294 (1990); Wu and Wallace, Gene, 4:560 (1989);Barringer, et al., Gene, 89:117 (1990), and Sooknanan and Malek,Biotechnology, 13:563-564 (1995). Improved methods of cloning in vitroamplified nucleic acids are described in Wallace, et al., U.S. Pat. No.5,426,039. Improved methods of amplifying large nucleic acids (up to 40kb) are summarized in Cheng, et al., Nature, 369:684-685 (1994) and thereferences therein. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausubel, Sambrook, Innis, and Berger, all supra.

One of skill will recognize many ways of generating alterations in agiven nucleic acid construct. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,Giliman and Smith, Gene 8:81-97 (1979), Roberts, et al., Nature,328:731-734 (1987) and Sambrook, Innis, Ausubel, Berger, and Mullis (allsupra).

Most modifications to nucleic acids are evaluated by routine screeningtechniques in suitable assays for the desired characteristic. Forinstance, changes in the immunological character of encoded polypeptidescan be detected by an appropriate immunological assay. For instance,changes in the cellular immunological character of the polypeptide canbe detected by an appropriate antibody or CTL assay. Modifications ofother properties such as nucleic acid hybridization to a complementarynucleic acid, redox or thermal stability of encoded proteins,hydrophobicity, susceptibility to proteolysis, or the tendency toaggregate are all assayed according to standard techniques.

A wide variety of formats and labels are available and appropriate fordetection of polypeptide sequences. These include analytic biochemicalmethods such as spectrophotometry, radiography, electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,and the like, and various immunological methods such as fluid or gelprecipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), western blot assays, immunofluorescentassays, and the like. Several commercially available ELISA assays forthe detection of retroviral components, including Env domains, areavailable, allowing one of skill to detect Env in biological samples.

Similarly, the detection of the nucleic acids of the present inventionproceeds by well known methods such as Southern analysis, northernanalysis, gel electrophoresis, PCR, radiolabeling and scintillationcounting, and affinity chromatography. Many assay formats areappropriate, including those reviewed in Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology—hybridization withnucleic acid probes parts I and II, Elsevier, N.Y. and Choo (ed) (1994)Methods In Molecular Biology Volume 33—In Situ Hybridization Protocols,Humana Press Inc., New Jersey (see also, other books in the Methods inMolecular Biology series); see especially, Chapter 21 of Choo (id.)“Detection of Virus Nucleic Acids by Radioactive and Nonisotopic in SituHybridization”. Finally, PCR is also routinely used to detect nucleicacids in biological samples (see, Innis, supra, for a generaldescription of PCR techniques).

In one preferred embodiment, antibodies are used to detect polypeptidesequences. Methods of producing polyclonal and monoclonal antibodies areknown to those of skill in the art, and many anti-HIV antibodies areavailable. See, e.g., Coligan (1991) Current Protocols in Immunology,Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A LaboratoryManual, Cold Spring Harbor Press, NY; Stites, et al. (eds.) Basic andClinical Immunology (4th ed.), Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.), Academic Press, New York,N.Y.; and Kohler and Milstein, Nature, 256:495-497 (1975). Othersuitable techniques for antibody preparation include selection oflibraries of recombinant antibodies in phage or similar vectors. See,Huse, et al., Science, 246:1275-1281 (1989); and Ward, et al., Nature,341:544-546 (1989). Specific monoclonal and polyclonal antibodies andantisera will usually bind with a KD of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most typically and preferably, 0.01 μM or better.

Development of HIV Env Vectors

To develop Env glycoprotein variants that might effectively inducehumoral and cellular immunity, a series of plasmid expression vectorswere generated (FIG. 178). HIV Env is encoded by nucleic acid sequencesthat contain RNA structures that limit gene expression. These vectorswere therefore synthesized using codons found in human genes that allowthese structures to be eliminated without affecting the amino acidsequence. Full-length HIV Env (gp160) was highly expressed in theabsence of HIV accessory proteins at levels ≧10-fold higher thanRev-dependent viral gp 160 in transfected 293 cell by Western blotanalysis (FIG. 179A), and the relevant mutant proteins were detected atthe expected apparent molecular weights (FIG. 179B-D). As might beanticipated, gp160 expressed from the synthetic gene was not efficientlyprocessed in transfected 293 cells, presumably because over-expressionof gp160 saturates the cellular proteases responsible for cleavage.(Binley J M et al., 2000, J. Virol, 74:627-643.) Synthetic HIV gp160induced toxicity in transfected cells, with cell rounding and detachmentevident within 48 hours (FIG. 180A vs B). This cytotoxicity was reducedby elimination of the COOH-terminal cytoplasmic domain. Env protein thatterminated at amino acid 752 (gp150) was less cytotoxic than gp160,while the shorter proteins (gp145 and gp140) produced little or noeffect (FIGS. 180C, D, and E, respectively).

To alter Env immunogenicity, two different approaches were explored.First the effects of glycosylation on cellular and humoral immunity wereevaluated by analysis of mutants in which conserved N-linkedglycosylation sites were eliminated by site-directed mutagenesis. Twosets of mutations were introduced into both gp160 and gp150 (FIG. 178).The first set included eleven potential sites (Δgly11), and the secondset included an additional 6 sites downstream (Δgly17). Expressionstudies showed that the glycosylation mutants were efficientlyexpressed, and the glycosylation mutant protein was appropriatelyreduced in size compared to wild type gp160 or gp150, consistent withreduced N-linked glycosylation (FIG. 179C).

The second approach involved a series of internal deletions designed tostabilize and expose functional domains of the protein that might bepresent in an extended helical structure prior to the formation of thesix-member coiled-coil structure in the hairpin intermediate. Togenerate this putative pre-hairpin structure, the cleavage site wasremoved to prevent the proteolytic processing of the envelope andstabilize the protein by linking it covalently to the gp41 extracellularand/or transmembrane domain. To reduce toxicity and enhance stability,the fusion peptide domain was deleted. The heptad repeats in theenvelope protein are important tertiary structure domains involved inthe ability of the envelope protein to form trimers. The sequencebetween the heptad repeats was removed to stabilize the formation oftrimers and eliminate formation of the hairpin intermediate. These ΔCFIdeletions were introduced into full-length gp160 and COOH-terminaltruncation mutants. Though cells transfected with vectors encodinggp140ΔCFI, gp145ΔCFI and gp160 readily expressed these proteins (FIG.179D), only gp140ΔCFI, which lacks the transmembrane domain, was readilydetected in the supernatant (FIG. 181), indicating that it can give riseto soluble antigen.

Immunogenicity of Env Mutants after DNA Vaccination

The ability of these Env proteins to elicit an immune response wasdetermined in mice by injection with these plasmid DNA expressionvectors. Antibody responses were monitored by the ability of antiserafrom injected mice to immunoprecipitate wild type gp160 from celllysates by SDS-PAGE and Western blotting (FIG. 182). In some cases,antibody reactivity was also confirmed by immunofluorescence. Toquantitate the antibody response, immunoprecipitation followed byWestern blotting with different dilutions of immunized mouse sera wastested. The intensity of the gp160 band was determined by densitometryand standardized relative to a positive control sera used to normalizedata between experiments. The approximately linear dose response ofgp160 intensity with serum dilution allowed quantification of theanti-gp160 antibody response in mouse sera. Data from immunized miceshowed that none of the wild type Env proteins, neither the gp160,gp150, gp145, nor gp140 COOH-terminal truncations generated consistentlyhigh antibody responses (FIG. 182A). Of these proteins, gp140 wassomewhat more effective than gp160 in generating antibody responses butremained low and inconsistent. Immunization with vectors designed toexpress glycosylation deficient envelope proteins also did not improvethe humoral response. In contrast, the ΔCFI mutants in the Envtruncation vectors substantially increased anti-gp160 antibody response(FIG. 182A; gp145, gp140, and gp128). The longer ΔCFI Env proteins(gp160 and gp150) did not show comparable enhancement of this response.The gp140 (ΔCFI) provided more consistent and a greater increase inantibody response than gp128ΔCFI (FIG. 182B). In all cases, thesevectors that encoded gp140ΔCFI, but not wild type gp140, inducedantibodies that were reactive to native gp160 (FIG. 182C).

To determine whether these modifications of Env adversely affected CTLresponses, spleen cells from immunized mice were tested for theirability to lyse relevant target cells. All mice immunized withcodon-altered Env vectors, including COOH-terminal deletion mutations(FIG. 183A) or glycosylation mutants (FIG. 183C), elicited strong CTLresponses directed to cell lines pulsed with HIV Env peptides. Thesefindings were also confirmed using stably transfected cells thatexpressed Env. Importantly, gp140 (ΔCFI), which elicited increasedantibody responses relative to the comparable wild typ Env, readilyinduced CTL responses to native Env, as did other ΔCFI mutants (FIG.183D). Addition of anti-CD8 antibody inhibited cytolytic activity, asdid depletion using magnetic beads coupled with anti-CD8 antibody, thusconfirming a cytotoxic T cell response to these immunogens after geneticimmunization (FIG. 183B). This response was detectable for at least sixmonths after immunization.

Glycosylation and ΔCFI HIV Env Mutants

To develop DNA vaccine candidates for HIV, we developed a series ofsynthetic genes designed to express HIV Env mutants in human cells. Inthe absence of HIV regulatory proteins, these codon-altered envelopeprotein genes expressed well in human cells. Like other DNA vaccines,immunization with these vectors elicited strong CTL responses in mice,and antibody responses were not robust in mice immunized with wild typeEnv expression vectors. Mutations in highly conserved N-linkedglycosylation sites did not significantly alter humoral or cellularimmune response to native Env. In contrast, a mutant Env with deletionsin the cleavage site, fusion domain, and a region between the heptadrepeats elicited a more potent humoral immune response and retained itsability to stimulate Env-specific CTL.

Recent reports suggest that gp160 forms trimers in vivo and the domainrequired for trimer formation resides in the ectodomain of the gp41.Such trimeric forms of HIV envelope protein are likely to presentdifferent epitopes to the immune system compared to monomeric gp120. Inaddition to the linear epitopes in the envelope, this trimeric structureis likely to expose conformational epitopes important for B celltriggering of a relevant antibody response. In this regard, gp140(ΔCFI), which induced the greatest antibody response, is released in asoluble form (FIG. 181). In contrast, wild type Env did not elicit hightiter antibody responses. The toxicity of Env in mammalian cells hasbeen seen and could limit both the amount and duration of envelopeprotein expression in vivo that would affect immunogenicity. Theenvelope is also heavily glycosylated, and removal of partial orcomplete gp120 glycosylation sites has resulted in higher titers ofstrain-specific neutralizing antibody responses to mutant SIVs inmonkeys. Though it seemed reasonable that deglycosylation would revealepitopes otherwise masked in the native protein, we did not observeenhanced immune reactivity by DNA vaccination using differentglycosylation site mutants, both in gp160 and gp150. This differencewith the previous study is likely due to the fact that DNA vaccinationrather than viral infection was utilized for immunization. Thoughglycosylation mutants are unlikely to prove helpful with this formermethod of immunization, we envision that modification of glycosylationsites will be effective with other vectors or adjuvants.

HIV-1 Env is proteolytically cleaved by a cellular convertase into gp120and gp41. The gp41 subunit is composed of cytoplasmic, transmembrane,and ectodomain segments. The role of the ectodomain of the envelope inmembrane fusion, particularly its hydrophobic glycine-rich fusionpeptide, is well established. Two regions with heptad coiled-coilrepeats in the ectodomain of gp41 are involved in viral fusion. Uponfusion, these two alpha helices, connected via a disulfide-stabilizedloop, presumably undergo a transient conformational change to a fusionactive state. These changes allow the formation of a six-member helicalhairpin intermediate structure that presumably exposes the fusionpeptide at the NH₂-terminus of gp41, allowing fusion to the target cellmembrane. The ΔCFI mutation was intended to eliminate cleavage of gp140,remove the unstable hydrophobic region and stabilize oligomer formation.Though detailed structural data is not yet available on this protein,these mutations apparently stabilize Env in a conformation that elicitsboth humoral and cellular immune responses. For example, theneutralizing epitope in the ectodomain of gp41 is present in the seriesof deletions and truncations of the envelope and gp140 (ΔCFI) isreactive with the 2F5 neutralizing monoclonal antibody that binds tothis epitope. Importantly, these immunogens also induced CTL responsesto Env. Though gp128 (ΔCFI) induced slightly more potent CTL activity,gp140 (ΔCFI) was better able to elicit such responses, both topeptide-pulsed cells and stably transduced target cells. Thus theenhanced humoral immune response introduced by this vaccine candidatedid not appear to diminish the CTL response. Taken together, theseresults indicate that gp140ΔCFI serves as an improved immunogen that canmore effectively elicit an antibody response against the envelope by DNAvaccination while preserving its ability to induce a CTL response.

Gag and Pol

In this disclosure, we have prepared synthetic HIV-1 B clade Gag and Polexpression vectors that are based on human (h) codon usage. Thesevectors encode hGag-Pol and its derivatives, hGag, hPol and an hGag-Polfusion protein. The synthetic Gag-Pol genes show little nucleotidehomology to HIV-1 but are the same in protein sequence. The modifiedGag-Pol genes were subcloned into a eukaryotic plasmid expression vectorfor expression and DNA immunization studies. Synthetic Gag-Pol genesallowed high level Rev-independent expression of HIV-1 Gag-Pol precursorproteins in human and mouse cell lines and induced significant cellularand humoral responses in mice. The Gag-Pol fusion protein induced thebroadest responses to Gag and Pol determinants and thus is envisioned asa prototype immunogen that maximizes epitope presentation.

Eliminating the Frame Shift Site to Create Viral Polyproteins

Described herein are HIV Gag-Pol fusion proteins encoded by a continuousopen reading frame so to improve the immune response to native Gag andPol. In some viruses, translational frame shifting is exploited duringprotein synthesis. Specific sequences in the RNA are required for theframe shifting. The viral RNA sequences cause ribosomal slippage so thatviral proteins are produced in non-equivalent ratios. For example,during translation in HIV, the ribosomes shift reading frames tosynthesize Gag precursor protein and the gag-pol fusion protein in a20:1 ratio. The strategy here is to maximize epitope presentation bytranscribing an immunogen from a continuous open reading frame byeliminating the frame shift site. Thus, the strategy for improving theimmune response by the use of a HIV Gag-Pol fusion protein encoded by acontinuous open reading frame extends to other viral proteins that areproduced in non-equivalent ratios by virtue of translational frameshifting.

The present invention involves HIV Gag-Pol fusion proteins encoded by asingle continuous open reading frame due to mutation of the frame shiftsite. The frame shift site is mutated by deletions, insertions, and/orsubstitutions of nucleotides to create a single continuous open readingframe. In one embodiment, deletions are meant (i.e., nucleotides aredeleted to create the same open reading frame).

The frame shift site is a mutated frame shift to create a singlecontinuous open reading frame. For example, a set of similar retroviralgag-pol frame shift sites are optionally made for a given fusionprotein, for example, by synthesizing different gag-pol frame shiftregions and cloning the sequences appropriately, or by site-directedmutagenesis of a given frame shift clone. The efficacy of the frameshift sites are assessed by measuring the production of the fusionprotein. The sequence that shows the highest level of expression is a“optimized” frame shift mutation for the set assessed. Alternatively,where a particular level of expression is desired, a frame shift sitefrom a particular set of possible frame shift sites which is closest tothe desired activity level is considered to be “optimized.”

Although a full length Gag sequence is preferred for use in the fusionprotein of the present invention, Gag is optionally deleted ofsubsequences without negating a polyepitope response. For example,regions of the matrix protein (p17), regions of the capsid protein(p24), regions of p2, regions of the nucleocapsid protein (p7), regionsof p1, and regions of p6 can be deleted while preserving the polyepitoperesponse. Alternatively, regions of the matrix protein (p17), regions ofthe capsid protein (p24), regions of p2, regions of the nucleocapsidprotein (p7), regions of p1, and regions of p6 can be substituted whilepreserving the polyepitope response. Alternatively, regions of thematrix protein (p17), regions of the capsid protein (p24), regions ofp2, regions of the nucleocapsid protein (p7), regions of p1, and regionsof p6 can be interrupted by insertions while preserving the polyepitoperesponse. Optionally, regions of the matrix protein (p17), regions ofthe capsid protein (p24), regions of p2, regions of the nucleocapsidprotein (p7), regions of p1, and regions of p6 can be mutated toinactivate these proteins.

Likewise, a full length Pol sequence is preferred for use in the fusionprotein of the present invention, and Pol is optionally deleted ofsubsequences without negating a polyepitope response. For example,regions of the protease protein, regions of the reverse transcriptaseprotein, and regions of the integrase protein can be deleted whilepreserving the polyepitope response. Alternatively, regions of theprotease protein, regions of the reverse transcriptase protein, andregions of the integrase protein can be substituted while preserving thepolyepitope response. Alternatively, regions of the protease protein,regions of the reverse transcriptase protein, and regions of theintegrase protein can be interrupted by insertions while preserving thepolyepitope response. Optionally, regions of the protease protein,regions of the reverse transcriptase protein, and regions of theintegrase protein can be mutated to inactivate these enzymes.

In other embodiments, the Gag-Pol fusion proteins are co-expressed withother proteins, either as fusion proteins or separately, preferably withEnv sequence proteins, such as the modified HIV Env ΔCFI proteinsdescribed herein.

In another aspect, the invention involves chimeric nucleic acidmolecules. The chimeric nucleic acid molecules of the inventiontypically have a retroviral gag nucleic acid sequence and a retroviralpol nucleic acid sequence in the same open reading frame due to mutationof the frame shift site. The continuous open reading frame encodes afusion protein such as those described above.

The chimeric nucleic acids of the present invention are optionally DNA,RNA, or mRNA. Most typically, the chimeric nucleic acids are provided byrecombinantly making a DNA, which is expressed in a cell as RNA and/oras mRNA. Given the strategy for making the chimeric nucleic acids of thepresent invention, one of skill can construct a variety of clonescontaining functionally equivalent nucleic acids. Cloning methodologiesto accomplish these ends, and sequencing methods to verify the sequenceof nucleic acids are well known in the art. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,volume 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook,et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,(Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel,et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel). Product information from manufacturers ofbiological reagents and experimental equipment also provide informationuseful in known biological methods. Such manufacturers include the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

The chimeric nucleic acid compositions of this invention, whether RNA,cDNA, mRNA, genomic DNA, or a hybrid of the various combinations, areisolated from biological sources or synthesized in vitro. The chimericnucleic acids of the present invention are present in transformed ortransfected whole cells, in transformed or transfected cell lysates, orin a partially purified or substantially pure form.

In vitro amplification techniques suitable for amplifying sequences toprovide a nucleic acid or for subsequent analysis, sequencing orsubcloning are known. Examples of techniques sufficient to directpersons of skill through such in vitro amplification methods, includingthe polymerase chain reaction (PCR) the ligase chain reaction (LCR),Qβ-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well asMullis, et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis, et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh, et al., Proc.Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli, et al., Proc. Natl.Acad. Sci. USA, 87:1874 (1990); Lomell, et al., J. Clin. Chem., 35:1826(1989); Landegren, et al., Science, 241:1077-1080 (1988); Van Brunt,Biotechnology, 8:291-294 (1990); Wu and Wallace, Gene, 4:560 (1989);Barringer, et al., Gene, 89:117 (1990), and Sooknanan and Malek,Biotechnology, 13:563-564 (1995). Improved methods of cloning in vitroamplified nucleic acids are described in Wallace, et al., U.S. Pat. No.5,426,039. Improved methods of amplifying large nucleic acids (up to 40kb) are summarized in Cheng, et al., Nature, 369:684-685 (1994) and thereferences therein. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausubel, Sambrook, Innis, and Berger, all supra.

One of skill will recognize many ways of generating alterations in agiven nucleic acid construct. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,Giliman and Smith, Gene 8:81-97 (1979), Roberts, et al., Nature,328:731-734 (1987) and Sambrook, Innis, Ausubel, Berger, and Mullis (allsupra).

Most modifications to nucleic acids are evaluated by routine screeningtechniques in suitable assays for the desired characteristic. Forinstance, changes in the immunological character of encoded polypeptidescan be detected by an appropriate immunological assay. For instance,changes in the cellular immunological character of the polypeptide canbe detected by an appropriate antibody or CTL assay. Modifications ofother properties such as nucleic acid hybridization to a complementarynucleic acid, redox or thermal stability of encoded proteins,hydrophobicity, susceptibility to proteolysis, or the tendency toaggregate are all assayed according to standard techniques.

A wide variety of formats and labels are available and appropriate fordetection of fusion protein sequences. These include analyticbiochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), western blot assays, immunofluorescentassays, and the like. Several commercially available ELISA assays forthe detection of retroviral components, including Env domains, areavailable, allowing one of skill to detect Env in biological samples.

Similarly, the detection of the chimeric nucleic acids of the presentinvention proceeds by well known methods such as Southern analysis,northern analysis, gel electrophoresis, PCR, radiolabeling andscintillation counting, and affinity chromatography. Many assay formatsare appropriate, including those reviewed in Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology—hybridization withnucleic acid probes parts I and II, Elsevier, N.Y. and Choo (ed) (1994)Methods In Molecular Biology Volume 33—In Situ Hybridization Protocols,Humana Press Inc., New Jersey (see also, other books in the Methods inMolecular Biology series); see especially, Chapter 21 of Choo (id.)“Detection of Virus Nucleic Acids by Radioactive and Nonisotopic in SituHybridization”. Finally, PCR is also routinely used to detect nucleicacids in biological samples (see, Innis, supra, for a generaldescription of PCR techniques).

In one preferred embodiment, antibodies are used to detect polypeptidesequences. Methods of producing polyclonal and monoclonal antibodies areknown to those of skill in the art, and many anti-HIV antibodies areavailable. See, e.g., Coligan (1991) Current Protocols in Immunology,Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A LaboratoryManual, Cold Spring Harbor Press, NY; Stites, et al. (eds.) Basic andClinical Immunology (4th ed.), Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.), Academic Press, New York,N.Y.; and Kohler and Milstein, Nature, 256:495-497 (1975). Othersuitable techniques for antibody preparation include selection oflibraries of recombinant antibodies in phage or similar vectors. See,Huse, et al., Science, 246:1275-1281 (1989); and Ward, et al., Nature,341:544-546 (1989). Specific monoclonal and polyclonal antibodies andantisera will usually bind with a KD of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most typically and preferably, 0.01 μM or better.

Expression of Synthetic HIV-1 Gag and Pol Genes

Four synthetic HIV-1 Gag-and/or Pol expression vectors, hGag-Pol,hGag-PolΔFsΔPr, hPol and hGag genes were prepared (FIG. 184). To confirmexpression, the synthetic or viral Gag-Pol genes were transientlytransfected into 293T cells, a human kidney-derived cell line. When celllysates were analyzed by immunoblotting with human anti-HIV-1 IgG (FIG.185A), monoclonal anti-p24 (FIG. 185B), and rabbit anti-RT (FIG. 185C),Gag p55, Pol p110 and Gag-Pol p160 precursor proteins were detected inhGag, hPol, and hGag-Pol fusion plasmids transfected 293T cells, as wasexpected. Mature virion proteins, p24 and RTp66, were detected in thehGag-Pol gene transfected cells (FIGS. 185A, B and C). This might be aresult of the activation of protease inside cells which was itself aresult of the high-level expression of Gag and Gag-Pol protein. Theexpression of Gag precursor proteins from codon-altered vectors was≧10-fold higher than viral Gag-Pol (FIG. 185), determined byquantitative phosphorimaging. The level of accumulated Gag-Pol fusionprotein was 100-fold higher in cells transfected with hGag-Pol comparedto viral Gag-Pol. Virus-like particles were released from the hGag genetransfected cells (FIG. 186), detected by transmission electronmicroscopy. Though such particles were observed at a lower frequencywith hGag-Pol, no particles were seen in cells transfected withhGag-PolΔFsΔPr or hPol vectors. Stable expression of HIV-1 Gag and Polproteins from codon-optimized genes in mouse CT26 and BH10ME cells wasalso observed (FIG. 185D).

Induction of HIV-1 Gag and Pol CTL Responses in Mice by DNA Vaccination

To evaluate the cellular immune response to HIV-1 Gag and Pol proteins,Balb/C female mice were injected intramuscularly with the eukaryoticexpression vector plasmids containing the codon-optimized genes. Twoweeks after the final vaccination, splenocytes were harvested from theimmunized mice and sensitized with either Gag or Pol peptide-pulsednaïve mouse splenocytes. One week later, CTL responses were analyzedusing a 5-hour chromium release assay.

CTL responses specific to HIV-1 Gag and/or Pol were first analyzed usingGag or Pol peptide-pulsed BC10ME cells, or mouse fibrosarcoma cell linesderived from B/C-N cells. Immunization with hGag, hGag-PolΔFSΔPr orhGag-Pol genes induced comparably strong CTL responses specific to Gag(FIG. 187A); however, after immunization with hPol, hGag-Pol ΔFSΔPr orhGag-Pol genes, only the fusion protein, hGag-PolΔFSΔPr, and hPol to alesser extent, elicited a marked CTL response to Pol (FIG. 187B). Toconfirm that the specific killing in the CTL assays was induced by CD8⁺cytotoxic T lymphocytes, CD4⁺ or CD8⁺ cells were depleted fromsensitized splenocytes by Dynal beads (Dynal, Inc., Lake Success, N.Y.).Depletion of CD8⁺ cells abolished the specific lysis in the hGag-PolΔFSΔPr gene-immunized mice, while depletion of CD4⁺ had little effect onlysis (FIG. 187C), suggesting that CD8⁺ lymphocytes were responsible forspecific cytotoxicity.

The responses were further analyzed and confirmed with the hGag or hPolgene transduced syngeneic CT26 and BC10ME cell lines. Responses to Gagin the mice immunized with the hGag, hGag-PolΔFSΔPr or hGag-Pol geneswere similar when peptide-pulsed cells were used as targets in the CTLassay (FIG. 188A). Mice immunized with the hPol gene generated aspecific response to HIV-1 Pol on BC10ME cell lines stably expressingPol as target cells (FIG. 188B). The same results have been observedwith CT26 cell lines. These stably transfected cell lines were thereforemore sensitive as target cells than peptide-pulsed cells in the Pol CTLassays.

Antibody Response in the Immunized Mice

Sera from mice immunized with different plasmids was analyzed with a p24ELISA. hGag immunized mice demonstrated the highest p24 antibody titers(FIG. 189A). Unexpectedly, hGag-Pol virus-like particles elicited thelowest levels of p24 antibody. Similar results were observed by Westernblotting with pooled sera (FIG. 189B). The HIV-1 Pol specific antibodieswere not detected by a commercially available Western blotting kit (FIG.189B), but antibodies to Pol were detected in mice immunized with hPoland hGag-PolΔFSΔPr with a more sensitive method, IP/Western blotting(FIG. 189C). Presumably, this assay is more sensitive and better able todetect native conformational epitopes. Though such antibodies were foundin mice immunized with Pol and Gag-Pol fusion proteins in this assay,minimal response was detected in the mice immunized with hGag-Pol.Though both immunogens elicited similar Gag responses, the Gag-Polfusion protein was therefore more effective in the stimulation of CTLand antibody responses to Pol.

HIV Gag-Pol Fusion Proteins

In this disclosure, HIV-1 B-clade Gag and Pol genes were modified toincrease Rev-independent expression of HIV-1 Gag-Pol proteins. Thismodification allowed synthesis of HIV Gag and Pol, as well as fusionproteins at levels 10- to 100-fold higher than the correspondingRev-dependent viral gene in the absence of Rev and RRE elements. Theseviral proteins were recognized by standard polyclonal and monoclonalantibodies (FIG. 185), and immature VLP were produced and released from293T cells transfected with hGag (FIG. 186).

The immune response induced by Rev-independent Gag-Pol was directedagainst both Gag and Pol determinants. The hGag, hGag-Pol fusion andhGag-Pol all induced strong CTL responses specific for Gag in miceimmunized with plasmid DNA, but a significant Pol response was elicitedonly in the mice immunized with the hGag-PolΔFSΔPr or Pol alone. Becauseimmunization with hGag-Pol gene failed to induce detectable cellular orhumoral responses to HIV-1 Pol protein, these findings indicate that theGag-Pol fusion protein induces a broader range of responses and allowsdelivery of an immunogen with a larger number of epitopes in a singlecontinuous open reading frame. During viral replication, viral gag-polproduces Gag precursor protein and the gag-pol fusion protein by frameshifting in a 20:1 ratio (Wilson, W et al., 1988, Cell, 55:1159-1169).The deletion of a frame shift site in hGag-PolΔFSΔPr results inproduction of only the Gag-Pol fusion protein. Expression of Gag-Polproteins alone in human cells is not adequate to form releasable viralparticles because HIV-1 viral assembly requires Gag precursor proteins(Park, J and C D Morrow, 1992, J Virol, 66:6304-6313; Smith, A J et al.,1993, J Virol, 67:2266-2275). The ability of hGag-PolΔFSΔPr to elicitstrong Gag and Pol specific CTL responses in mice may be explained byhigh level expression of Gag-Pol fusion protein and its retention on theinside of cells, which could create a new condition not existing duringnormal viral replication and could provide sufficient proteins forantigen presentation. Moreover, the mutation in viral protease preventsviral protein from intracellular activation and reduced cellulartoxicity. Overexpression of this polyprotein is also likely to affectits intracellular localization/transport and is envisioned as improvingantigen presentation.

The Pol gene of HIV-1 is the precursor protein for viral protease,reverse transcriptase and integrase, which are crucial to viralreplication (Kohl, N E et al., 1988, Proc. Natl. Acad. Sci. USA,85:4686-4690.). Retroviral extracellular maturation, resulting fromself-activation of protease after release produces mature RT and INwhich have important functions in reverse transcription and integrationrespectively, for HIV-1, HIV-2, and SIV replication. The catalytic coresof these enzymes have relatively conserved domains in order to preservetheir functions and thus are envisioned as inducing cross-clade CTLresponses. As early as 1988, CTLs specific for HIV-1 RT were found inblood samples from HIV-1 infected individuals (Hosmalin, A et al., 1990,Proc. Natl. Acad. Sci. USA, 87:2344-2348; Walker, B D et al., 1988,Science, 240:64-66.). Relatively strong Gag-specific CTL responses havebeen shown in numerous non-human primate and human studies, using DNAvaccines or a live recombinant vector containing viral Gag-Polconstructs (Evans, T G, 1999, J Infect Dis, 180:290-298; Ferrari, G,1997, Blood, 90:2406-2416; Gorse, G J et al., 1999, Vaccine, 18:835-849;Seth, A, 1998, Proc. Natl. Acad. Sci. USA, 95:10112-10116; Seth, A etal., 2000, J Virol, 74:2502-2509), but fewer Pol-specific CTL responseshave been reported. The detection of significant CTL responses specificto Pol in our disclosure may be attributed in part to establishment ofstable Pol expressing cell lines, in which codon alteration andinactivation of FS and PR in the Pol gene allow high level expression ofthe Pol protein without cellular toxicity. Though it remains possiblethat the hGag-Pol, or a combination of hGag and hPol, may exert similareffects with appropriate adjuvants or with different prime-boostregimens, the Rev-independent Gag-Pol fusion protein stimulates HIV-1Gag and Pol specific CTL responses in mice and is envisioned as provinguseful in an AIDS vaccine.

Plasmid Descriptions Env Plasmids VRC2100

pVR1012x/s R5gp139-Nef (Delta) MHC (Delta) CD4/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The envelope protein gene frompX4gp160/h (Nabel lab #1272) was ligated in frame with the mutant Nefgene from pNefDMHCDCD4/h (Nabel lab #1278) to producepX4gp139-NefDMHCDCD4/h. The envelope-Nef fusion protein expressed frompX4gp139-NefDMHCDCD4/h contains the first 668 amino acids from the HIVenvelope glycoprotein (gp139) fused to the entire mutant Nef protein.The truncated envelope polyprotein (gp139) contains the entire SUprotein and a portion of the TM protein including the fusion domain, butlacking the transmembrane domain and regions important for oligomerformation. The protein sequence of the Nef protein from HIV-1 PV22(GenBank accession number K02083) was used to create a synthetic versionof the Nef gene (Nef/h) using codons optimized for expression in humancells. To disrupt the ability of Nef to limit both MHC class I and CD4expression, point mutations were introduced into the Nef gene frompNef/h (Nabel lab #1275). The resulting amino acids substitutions inpNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174A and D175A.X4gp139-NefDMHCDCD4/h is expressed from the pVR1012x/s (Nabel lab #1267) vector backbone.

VRC2200

pVR1012x/s R5gp157-Nef (Delta) MHC (Delta) CD4/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (Nabel lab number1272) were replaced with the corresponding region from the BaL strain ofHIV-1 (GeneBank accession number M68893, again using human preferredcodons). The envelope-Nef fusion protein expressed from pR5gp157-Nef/hcontains the first 820 amino acids from the HIV envelope glycoprotein(gp157) fused to the entire mutant Nef protein. The gene for gp157 wasligated in frame with the full-length mutant Nef gene frompNefDMHCDCD4/h (Nabel lab #1278) to produce pR5gp157-NefDMHCDCD4/h. Theprotein sequence of the Nef protein from HIV-1 PV22 (GenBank accessionnumber K02083) was used to create a synthetic version of the Nef gene(Nef/h) using codons optimized for expression in human cells. To disruptthe ability of Nef to limit both MHC class I and CD4 expression, pointmutations were introduced into the Nef gene from pNef/h (Nabel lab#1275). The resulting amino acids substitutions in pNefDMHCDCD4/h are:P69A, P72A, P75A, P78A, D174A and D175A. R5gp157-NefDMHCDCD4/h isexpressed from the pVR1102x/s (Nabel lab #1267) vector backbone.

VRC2300

pVR1012x/s X4gp139-Nef deltaMHC deltaCD4/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The envelope protein gene frompX4gp160/h (Nabel lab #1272) was ligated in frame with the mutant Nefgene from pNefDMHC/h (Nabel lab #1276) to produce pX4gp139-NefDMHC/h.The envelope-Nef fusion protein expressed from pX4gp139-NefDMHC/hcontains the first 668 amino acids from the HIV envelope glycoprotein(gp139) fused to the entire mutant Nef protein. The truncated envelopepolyprotein (gp139) contains the entire SU protein and a portion of theTM protein including the fusion domain, but lacking the transmembranedomain and regions important for oligomer formation. The proteinsequence of the Nef protein from HIV-1 PV22 (GenBank accession numberK02083) was used to create a synthetic version of the Nef gene (Nef/h)using codons optimized for expression in human cells. To disrupt theability of Nef to limit MHC class I expression, point mutations wereintroduced into the Nef gene from pNef/h (Nabel lab #1275). Theresulting amino acids substitutions in pNefDMHC/h are: P69A, P72A, P75A,and P78A. X4gp139-NefDMHC/h is expressed from the pVR1012x/s (Nabel lab#1267) vector backbone

VRC2302

pVR1012x/s X4gp130-Nef/h

For the X4gp130/h (VRC2703) portion, the protein sequence of theenvelope polyprotein (gp160) from HXB2 (X4-tropic, GenBank accessionnumber K03455) was used to create a synthetic version of the gene(X4gp160/h) using codons optimized for expression in human cells. Thenucleotide sequence X4gp160/h shows little homology to the HXB2 gene,but the protein encoded is the same with the following amino acidsubstitutions: F53L, N94D, K192S, I215N, A224T, A346D, and P470L. Thefull-length X4-tropic version of the envelope protein from pX4gp160/h(VRC3300) was terminated after the codon for amino acid 602. Thetruncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking thetransmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. Regions important for oligomer formationmay be partially functional. This X4 gp130/h (VRC2703) gene was fused toNef gene. The Nef protein from HIV-1 PV22 (GenBank accession numberK02083) was used to create the viral Nef gene (pVR1012-Nef)(Nabel Lab#1093. The nucleotide sequence is homologous to the viral gene, and theprotein encoded is the same. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2400

pVR1012x/s X4gp157-NefDMHCDCD4/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The envelope-Nef fusion proteinexpressed from pX4gp157-NefDMHCDCD4/h contains the first 820 amino acidsfrom the HIV envelope glycoprotein (gp157) fused to the entire mutantNef protein. The truncated envelope polyprotein (gp157) contains theentire SU protein and a portion of the TM protein including the fusiondomain, the transmembrane domain and regions important for oligomerformation. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The gene for gp157 was ligated in frame with thefull-length mutant Nef gene from pNefDMHCDCD4/h (VRC3600) to producepX4gp157-NefDMHCDCD4/h. The protein sequence of the Nef protein fromHIV-1 PV22 (GenBank accession number K02083) was used to create asynthetic version of the Nef gene (Nef/h) using codons optimized forexpression in human cells. To disrupt the ability of Nef to limit bothMHC class I and CD4 expression, point mutations were introduced into theNef gene from pNef/h (VRC3500). The resulting amino acids substitutionsin pNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174A and D175A.X4gp160-NefDMHCDCD4/h is expressed from the pVR1012x/s (VRC2000) vectorbackbone.

VRC2700

pVR1012x/s X4gp140/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The full-length X4-tropic versionof the envelope protein from pX4gp160/h (VRC3300) was terminated afterthe codon for amino acid 680. The truncated envelope polyproteincontains the entire SU protein and a portion of the TM protein includingthe fusion domain, but lacking the transmembrane domain. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC2701

pVR1012x/s X4gp140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. The full-length X4-tropic version of theenvelope protein from pX4gp160/h (VRC3300) was terminated after thecodon for amino acid 680. The truncated envelope polyprotein containsthe entire SU protein and a portion of the TM protein including thefusion domain, but lacking the transmembrane domain. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2702

pVR1012x/s X4gp128(del F/CL)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. The full-length X4-tropic version of theenvelope protein from pX4gp160/h (VRC3300) was terminated after thecodon for amino acid 592. The truncated envelope polyprotein containsthe entire SU protein and a portion of the TM protein including thefusion domain, but lacking the transmembrane domain. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC2706

pVR1012x/s X4gp 145/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. The full-length X4-tropic version of theenvelope protein from pX4gp160/h (VRC3300) was terminated after thecodon for amino acid 704. The truncated envelope polyprotein containsthe entire SU protein and a portion of the TM protein including thefusion domain, but lacking the transmembrane domain. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC2707

pVR1012x/s X4gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. The full-length X4-tropic version of theenvelope protein from pX4gp160/h (VRC3300) was terminated after thecodon for amino acid 704. The truncated envelope polyprotein containsthe entire SU protein and a portion of the TM protein including thefusion domain, but lacking the transmembrane domain. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2800

pVR1012x/s R5gp 140/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking thetransmembrane domain. Regions important for oligomer formation may bepartially functional. The expression vector backbone is pVR1012x/s(VRC2000).

VRC2801

pVR1012x/s R5gp140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking thetransmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 503-536, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 593-620, have been deleted. Regionsimportant for oligomer formation may be partially functional. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC2804

pVR1012x/s R5gp145/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC2805

pVR1012x/s R5gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheFusion and Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2810

pVR1012 x/s R5gp140delC1 (delCFI)/h

Constant domain 1 was deleted from gp140delCFI from amino acid 33-127and was replaced with a NheI site.

VRC2811

pVR1012x/s R5gp140delC2 (delCFI)/h

Constant domain 2 was deleted from gp140delCFI from amino acid 199-293,and was replaced with an NheI site

VRC2812

pVR1012x/s R5gp140delC3 (delCFI)/h

Constant domain 3 was deleted from gp140delCFI from amino acid 333-380,and was replaced with an NheI site.

VRC2813

pVR1012x/s R5gp140delC4 (delCFI)/h

Constant domain 4 was deleted from gp140delCFI from amino acid 419-458,and was replaced with an NheI site.

VRC2814

pVR1012x/s R5gp140delC5 (delCFI)/h

Constant domain 5 was deleted from gp140delCFI from amino acid 472-498and was replaced with an NheI site.

VRC2820

pVR1012x/s R5gp 140(dCFI)/dV1

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1 loop (a.a.129 to 154) and transmembrane domain. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2821

pVR1012x/s R5gp 140(dCFI)/dV2

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2 loop (a.a.160 to 193) and transmembrane domain. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispVR1012x/s (VRC2000).

VRC2822

pVR1012x/s R5gp140(dCFI)/dV3

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV3 loop (a.a.299 to 327) and transmembrane domain. Heptad (H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispVR1012x/s (VRC2000).

VRC 2823

pVR1012x/s R5gp 140(dCFI)/dV4

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV4 loop (a.a.386 to 413) and transmembrane domain. Heptad (H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispVR1012x/s (VRC2000).

VRC 2824

pVR1012x/s R5gp 140(dCFI)/dV12

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2 loops (a.a.129 to 154, and a.a.160 to 193) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2825

pVR1012x/s R5gp 140(dCFI)/dV13

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V3 loops (a.a.129 to 154, and a.a.299 to 327) and transmembranedomain. Heptad (H) 1, Heptad 2 and their Interspace (IS) are requiredfor oligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2826

pVR1012x/s R5gp 140(dCFI)/dV14

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V4 loops (a.a.129 to 154, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2827

pVR1012x/s R5gp140(dCFI)/dV23

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V3 loops (a.a.160 to 193, and a.a.299 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2828

pVR1012x/s R5gp140(dCFI)/dV24

The protein sequence of the envelope polyprotein (gp 160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V4 loops (a.a.160 to 193, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2829

pVR1012x/s R5gp 140(dCFI)/dV34

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV3, V4 loops (a.a.299 to 327, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pVR1012x/s (VRC2000).

VRC 2830

pVR1012x/s R5gp 140(dCFI)/dV123

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V3 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.299 to 327)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2831

pVR1012x/s R5gp 140(dCFI)/dV124

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V4 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.386 to 413)and transmembrane domain. Heptad (H) 1, Heptad 2 and their Interspace(IS) are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2832

pVR1012x/s R5gp140(dCFI)/dV134

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V3, V4 loops (a.a. 129 to 154, a.a.299 to 327, and a.a.386 to 413)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2833

pVR1012x/s R5gp 140(dCFI)/dV234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V3, V4 loops (a.a. 160 to 193, a.a.299 to 327, and a.a.386 to 413)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2834

pVR1012x/s R5gp 140(dCFI)/dV1234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V3, V4 loops (a.a. 129 to 154, a.a. 160 to 193, a.a.299 to 327,and a.a.386 to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 andtheir Interspace(IS) are required for oligomerization. The Fusion andCleavage (F/CL) domains, from amino acids 503-536, have been deleted.The Interspace (IS) between Heptad (H) 1 and 2, from amino acids593-620, have been deleted. Regions important for oligomer formation maybe partially functional. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 2835

pAdApt R5gp140(dCFI)/dV1

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1 loop (a.a.129 to 154) and transmembrane domain. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispAdApt.

VRC 2836

pAdApt R5gp140(dCFI)/dV2

The protein sequence of the envelope polyprotein (gp 160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2 loop (a.a. 160 to 193) and transmembrane domain. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone isAdApt.

VRC 2837

pAdApt R5gp140(dCFI)/dV3

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV3 loop (a.a.299 to 327) and transmembrane domain. Heptad (H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispAdApt.

VRC 2838

pAdApt R5gp140(dCFI)/dV4

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV4 loop (a.a.386 to 413) and transmembrane domain. Heptad (H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. The Fusionand Cleavage (F/CL) domains, from amino acids 503-536, have beendeleted. The Interspace (IS) between Heptad (H) 1 and 2, from aminoacids 593-620, have been deleted. Regions important for oligomerformation may be partially functional. The expression vector backbone ispAdApt.

VRC 2839

pAdApt R5gp 140(dCFI)/dV12

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2 loops (a.a.129 to 154, and a.a.160 to 193) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2840

pAdApt R5gp 140(dCFI)/dV13

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V3 loops (a.a.129 to 154, and a.a.299 to 327) and transmembranedomain. Heptad (H) 1, Heptad 2 and their Interspace (IS) are requiredfor oligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2841

pAdApt R5gp 140(dCFI)/dV14

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V4 loops (a.a.129 to 154, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2842

pAdApt R5gp140(dCFI)/dV23

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V3 loops (a.a.160 to 193, and a.a.299 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2843

pAdApt R5gp140(dCFI)/dV24

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V4 loops (a.a.160 to 193, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2844

pAdApt R5gp140(dCFI)/dV34

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV3, V4 loops (a.a.299 to 327, and a.a.386 to 413) and transmembranedomain. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. Regions importantfor oligomer formation may be partially functional. The expressionvector backbone is pAdApt.

VRC 2845

pAdApt R5gp 140(dCFI)/dV123

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V3 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.299 to 327)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pAdApt

VRC 2846

pAdApt R5gp140(dCFI)/dV124

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V4 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.386 to 413)and transmembrane domain. Heptad (H) 1, Heptad 2 and their Interspace(IS) are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pAdApt.

VRC 2847

pAdApt R5gp 140(dCFI)/dV134

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V3, V4 loops (a.a. 129 to 154, a.a.299 to 327, and a.a.386 to 413)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pAdApt.

VRC 2848

pAdApt R5gp 140(dCFI)/dV234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV2, V3, V4 loops (a.a. 160 to 193, a.a.299 to 327, and a.a.386 to 413)and transmembrane domain. Heptad(H) 1, Heptad 2 and their Interspace(IS)are required for oligomerization. The Fusion and Cleavage (F/CL)domains, from amino acids 503-536, have been deleted. The Interspace(IS) between Heptad (H) 1 and 2, from amino acids 593-620, have beendeleted. Regions important for oligomer formation may be partiallyfunctional. The expression vector backbone is pAdApt.

VRC 2849

pAdApt R5gp 140(dCFI)/dV1234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated envelope polyprotein contains the entire SU protein and aportion of the TM protein including the fusion domain, but lacking theV1, V2, V3, V4 loops (a.a. 129 to 154, a.a. 160 to 193, a.a.299 to 327,and a.a.386 to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 andtheir Interspace(IS) are required for oligomerization. The Fusion andCleavage (F/CL) domains, from amino acids 503-536, have been deleted.The Interspace (IS) between Heptad (H) 1 and 2, from amino acids593-620, have been deleted. Regions important for oligomer formation maybe partially functional. The expression vector backbone is pAdApt.

VRC2850

pVR1012x/s R5gp145delC1 (delCFI)/h

Constant domain 1 was deleted from gp145delCFI from amino acid 33-127and was replaced with an Nhe I site.

VRC2851

pVR1012 x/s R5gp145delC2 (delCFI)/h

Constant domain 2 was deleted from gp145dCFI from amino acid 199-293 andwas replaced with an Nhe I site.

VRC2852

pVR1012x/s R5gp145delC3 (delCFI)/h

Constant domain 3 was deleted from gp145delCFI from amino acid 333-380and was replaced with an Nhe I site.

VRC2853

pVR1012 x/s R5gp145delC4 (delCFI)/h

Constant domain 4 was deleted from gp145delCFI from amino acid 419-458and was replaced with an Nhe I site.

VRC2854

pVR1012 x/s R5gp145delC5 (delCFI)/h

Constant domain 5 was deleted from gp145delCFI from amino acid 472-498and was replaced with an Nhe I site.

VRC 2860

pVR1012x/s R5gp145(dCFI)/h/dV1

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheV1 loop (a.a. 129-154) and Fusion and Cleavage (F/CL) domains (a.a.503-536) have been deleted. Also, the Interspace (IS) between Heptad (H)1 and 2 (a.a.593-620) have been deleted. The expression vector backboneis pVR1012x/s (VRC2000).

VRC 2861

pVR1012x/s R5gp145(dCFI)/h/dV2

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheV2 loop (a.a. 160-193) and Fusion and Cleavage (F/CL) domains (a.a.503-536) have been deleted. Also, the Interspace (IS) between Heptad (H)1 and 2 (a.a.593-620) have been deleted. The expression vector backboneis pVR1012x/s (VRC2000).

VRC 2862

pVR1012x/s R5gp145(dCFI)/h/dV3

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheV3 loop (a.a. 299-327) and Fusion and Cleavage (F/CL) domains (a.a.503-536) have been deleted. Also, the Interspace (IS) between Heptad (H)1 and 2 (a.a.593-620) have been deleted. The expression vector backboneis pVR1012x/s (VRC2000).

VRC 2863

pVR1012x/s R5gp145(dCFI)/h/dV4

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV4 loop (a.a. 386-413) and Fusion and Cleavage (F/CL) domains (a.a.503-536) have been deleted. Also, the Interspace (IS) between Heptad (H)1 and 2 (a.a.593-620) have been deleted. The expression vector backboneis pVR1012x/s (VRC2000).

VRC 2864

pVR10I2x/s R5gp145(dCFI)/h/dV12

The protein sequence of the envelope polyprotein (gp 160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheV1, V2 loops (a.a. 129-154 and 160-193) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR0102x/s (VRC2000).

VRC 2865

pVR1012x/s R5gp145(dCFI)/h/dV13

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V3 loops (a.a. 129-154 and 299-327) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 2866

pVR1012x/s R5gp145(dCFI)/h/dV14

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V4 loops (a.a. 129-154 and 386-413) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 2867

pVR1012x/s R5gp145(dCFI)/h/dV23

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV2, V3 loops (a.a. 160-193 and 299-327) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 2868

pVR1012x/s R5gp145(dCFI)/h/dV24

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad(H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV2, V4 loops (a.a. 160-193 and 386-413) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 2869

pVR1012x/s R5gp 145(dCFI)/h/dV34

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV3, V4 loops (a.a. 299-327 and 386-413) and Fusion and Cleavage (F/CL)domains (a.a. 503-536) have been deleted. Also, the Interspace (IS)between Heptad (H) 1 and 2 (a.a.593-620) have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 2870

pVR1012x/s R5gp145(dCFI)/h/dV134

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V3, V4 loops (a.a. 129-154, 299-327 and 386-413) and Fusion andCleavage (F/CL) domains (a.a. 503-536) have been deleted. Also, theInterspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have beendeleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2871

pVR1012x/s R5gp 145(dCFI)/h/dV234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace(IS) are required for oligomerization. TheV2, V3, V4 loops (a.a. 160-193, 299-327 and 386-413) and Fusion andCleavage (F/CL) domains (a.a. 503-536) have been deleted. Also, theInterspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have beendeleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2872

pVR1012x/s R5gp145(dCFI)dv123/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V2, V4 loops (a.a. 129-154, 160-193 and 386-413) and Fusion andCleavage (F/CL) domains (a.a. 503-536) have been deleted. Also, theInterspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have beendeleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2873

pVR1012x/s R5gp 145(dCFI)/h/dV124

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V2, V4 loops (a.a. 129-154, 160-193 and 386-413) and Fusion andCleavage (F/CL) domains (a.a. 503-536) have been deleted. Also, theInterspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have beendeleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC 2874

pVR1012x/s R5gp 145(dCFI)/h/dV1234

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, and P470L. To produce an R5-tropic version of the envelopeprotein (R5gp160/h), the region encoding HIV-1 envelope polyproteinamino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with thecorresponding region from the BaL strain of HIV-1 (GeneBank accessionnumber M68893, again using human preferred codons). The full-lengthR5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000)was terminated after the codon for amino acid 704. The truncatedenvelope polyprotein (gp145) contains the entire SU protein and aportion of the TM protein including the fusion domain, the transmembranedomain, and regions important for oligomer formation. Heptad (H) 1,Heptad 2 and their Interspace (IS) are required for oligomerization. TheV1, V2, V3, V4 loops (a.a. 129-154, 160-193, 299-327 and 386-413) andFusion and Cleavage (F/CL) domains (a.a. 503-536) have been deleted.Also, the Interspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) havebeen deleted. The expression vector backbone is pVR1012x/s (VRC2000).

VRC2900

pVR1012x/s R5gp150/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The full-length R5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 752.The truncated envelope polyprotein (gp150) contains the entire SUprotein and a portion of the TM protein including the fusion domain, thetransmembrane domain, and regions important for oligomer formation. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC3000

pVR1012x/s R5gp 160/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).Full length SU and TM proteins are expressed from the pVR1012x/s(VRC2000) vector backbone.

VRC3200

pVR1012x/s X4gp150/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The full-length X4-tropic versionof the envelope protein gene from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 752. The truncated envelope polyprotein(gp150) contains the entire SU protein and a portion of the TM proteinincluding the fusion domain, the transmembrane domain, and regionsimportant for oligomer formation. The expression vector backbone ispVR1012x/s (VRC2000).

VRC3201

pVR1012x/s X4gp150(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. The full-length X4-tropic versionof the envelope protein gene from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 752. The truncated envelope polyprotein(gp150) contains the entire SU protein and a portion of the TM proteinincluding the fusion domain, the transmembrane domain, and regionsimportant for oligomer formation. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 503-536, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 593-620,have been deleted. The expression vector backbone is pVR1012x/s(VRC2000).

VRC3202

pVR1012x/s X4gp150 Δgly/h.

Eukaryotic vector with humanized codons expressing the HIV envelopeglycoprotein gp150 from HXB2, X4 tropic mutated in the Glycosylationsites. VRC3202 pVR1012x/s X4gp150Dgly/h The protein sequence of theenvelope polyprotein (gp160) from HXB2 (X4-tropic, GenBank accessionnumber K03455) was used to create a synthetic version of the gene(X4gp160/h) using codons optimized for expression in human cells. Thenucleotide sequence X4gp160/h shows little homology to the HXB2 gene,but the protein encoded is the same with the following amino acidsubstitutions: F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, andS745T. To disrupt potential glycoslylation sites in the HIV-1 envelopeproteins, point mutations were introduced into the full-length X4-tropicversion of the envelope protein gene from pX4gp160/h (VRC3300). Theresulting amino acids substitutions in X4gp160Dgly/h are: N88D, N156D,N160D, N197E, N230D, N234D, N241D, N276D, L288V, N289D, S291T, N295D,N332D, N339D, N356D, N386D, and N448D. The full-length X4-tropic versionof the envelope protein gene from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 752. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. Full length SU and TMproteins are expressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3203

pVR1012x/s X4gp 150 AB Δgly/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The AB designation means the amino acids from1-307 (XbaI to EcoR1). The nucleotide sequence X4gp160/h shows littlehomology to the HXB2 gene, but the protein encoded is the same with thefollowing amino acid substitutions: F53L, N94D, K192S, I215N, A224T,A346D, P470L, T7231, and S745T. To disrupt potential glycoslylationsites in the HIV-1 envelope proteins, point mutations were introducedinto the full-length X4-tropic version of the envelope protein gene frompX4gp160/h (VRC3300). The resulting amino acids substitutions inX4gp160Dgly/h are: N88D, N156D, N160D, N197E, N230D, N234D, N241D,N276D, L288V, N289D, S291T, and N295D. The full-length X4-tropic versionof the envelope protein gene from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 752. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. Full length SU and TMproteins are expressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3300

pVR1012x/s X4gp 160/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. Full length SU and TMproteins are expressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3301

pVR1012x/s X4gp160(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. Full length SU and TM proteinsare Expressed. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 503-536, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 593-620, have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC3400

pVR1012x/s X4gp160Δgly/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To disrupt potentialglycoslylation sites in the HIV-1 envelope proteins, point mutationswere introduced into the full-length X4-tropic version of the envelopeprotein gene from pX4gp160/h (VRC3300). The resulting amino acidssubstitutions in X4gp160Dgly/h are: N88D, N156D, N160D, N197E, N230D,N234D, N241D, N276D, L288V, N289D, S291T, N295D, N332D, N339D, N356D,N386D, and N448D. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. Full length SU and TM proteins areexpressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3401

pVR1012x/s X4gp 160AB mut Δgly/h

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The AB designation means the amino acids from1-307 (XbaI to EcoR1). The nucleotide sequence X4gp160/h shows littlehomology to the HXB2 gene, but the protein encoded is the same with thefollowing amino acid substitutions: F53L, N94D, K192S, I215N, A224T,A346D, P470L, T7231, and S745T. To disrupt potential glycoslylationsites in the HIV-1 envelope proteins, point mutations were introducedinto the full-length X4-tropic version of the envelope protein gene frompX4gp160/h (VRC3300). The resulting amino acids substitutions inX4gp160Dgly/h are: N88D, N156D, N160D, N197E, N230D, N234D, N241D,N276D, L288V, N289D, S291T, and N295D. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. Full length SU and TMproteins are expressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3500

pVR1012x/s Nef/h

The protein sequence of the Nef protein from HIV-1 PV22 (GenBankaccession number K02083) was used to create a synthetic version of theNef gene (Nef/h) using codons optimized for expression in human cells.The nucleotide sequence Nef/h shows little homology to the viral gene,but the protein encoded is the same. Nef/h is expressed from thepVR1012x/s (VRC2000) vector backbone.

VRC3600

pVR1012x/s NefDMHCDCD4/h

The protein sequence of the Nef protein from HIV-1 PV22 (GenBankaccession number K02083) was used to create a synthetic version of theNef gene (Nef/h) using codons optimized for expression in human cells.To disrupt the ability of Nef to limit both MHC class I and CD4expression point mutations were introduced into the Nef gene from pNef/h(VRC3500). The resulting amino acids substitutions in pNefDMHCDCD4/hare: P69A, P72A, P75A, P78A, D174A and D175A. pNefDMHCDCD4/h isexpressed from the pVR1012x/s (VRC2000) vector backbone.

VRC3700

pVR1012x/s NefDCD4/h

The protein sequence of the Nef protein from HIV-1 PV22 (GenBankaccession number K02083) was used to create a synthetic version o f theNef gene (Nef/h) using codons optimized for expression in human cells.To disrupt the ability of Nef to limit CD4 expression, point mutationswere introduced into the Nef gene from pNef/h (VRC3500). The resultingamino acids substitutions in pNefDCD/h are: D174A and D175A. pNefDCD4/his expressed from the pVR1012x/s (VRC2000) vector backbone. VRC3700pVR1012x/s NefDCD4/h The protein sequence of the Nef protein from HIV-1PV22 (GenBank accession number K02083) was used to create a syntheticversion of the Nef gene (Nef/h) using codons optimized for expression inhuman cells. To disrupt the ability of Nef to limit CD4 expression,point mutations were introduced into the Nef gene from pNef/h (VRC3500).The resulting amino acids substitutions in pNefDCD/h are: D174A andD175A. pNefDCD4/h is expressed from the pVR1012x/s (VRC2000) vectorbackbone.

VRC3800

pVR1012x/s NefDMHC/h

The protein sequence of the Nef protein from HIV-1 PV22 (GenBankaccession number K02083) was used to create a synthetic version of theNef gene (Nef/h) using codons optimized for expression in human cells.To disrupt the ability of Nef to limit MHC class I expression, pointmutations were introduced into the Nef gene from pNef/h (VRC3500). Theresulting amino acids substitutions in pNefDMHC/h are: P69A, P72A, P75A,and P78A. pNefDMHC/h is expressed from the pVR1012x/s (VRC2000) vectorbackbone.

VRC5200

pVR1012x/s 89.6 Pgp128(del F/CL)/h

The protein sequence of the envelope polyprotein (gp160) from 89.6P(Dual-tropic, GenBank accession number u89134/LOCUS:SIU89134) was usedto create a synthetic version of the gene (89.6 Pgp160/h) using codonsoptimized for expression in human cells. The nucleotide sequence 89.6Pgp160/h shows little homology to the 89.6P gene, but the proteinencoded is the same. The full-length 89.6P, dual-tropic version of theenvelope protein gene from 89.6P gp160/h (VRC3000) was terminated afterthe codon for amino acid 596. The truncated envelope polyproteincontains the entire SU protein and a portion of the TM protein includingthe fusion domain, but lacking the transmembrane domain. The Fusion andCleavage (F/CL) domains, from amino acids 508-541, have been deleted.The expression vector backbone is pVR1012x/s (VRC2000).

VRC5201

pVR1012x/s 89.6 Pgp140(del F/CL del H IS/h

The protein sequence of the envelope polyprotein (gp160) from 89.6P(dual-tropic, GenBank accession number u89134/locus SIU89134) was usedto create a synthetic version of the gene (Dualtropic gp160/h) usingcodons optimized for expression in human cells. The nucleotide sequencedualtropic gp160/h shows little homology to the 89.6P gene, but theprotein encoded is the same. The full-length 89.6P, dual-tropic versionof the envelope protein gene was terminated after the codon for aminoacid 683. The truncated envelope polyprotein contains the entire SUprotein and a portion of the TM protein including the fusion domain, butlacking the transmembrane domain. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 508-541, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 597-625,have been deleted. Regions important for oligomer formation may bepartially functional. The expression vector backbone is pVR1012x/s(VRC2000).

VRC5202

pVR1012x/s 89.6 Pgp145(del F/CL del H IS/h

The protein sequence of the envelope polyprotein (gp160) from 89.6P(dual-tropic, GenBank accession number u89134/locus SIU89134) was usedto create a synthetic version of the gene (Dualtropic gp 160/h) usingcodons optimized for expression in human cells. The nucleotide sequencedualtropic gp160/h shows little homology to the 89.6P gene, but theprotein encoded is the same. The full-length 89.6P, dual-tropic versionof the envelope protein gene was terminated after the codon for aminoacid 709. The truncated envelope polyprotein contains the entire SUprotein and a portion of the TM protein including the fusion domain, butlacking the transmembrane domain. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 508-541, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 597-625,have been deleted. Regions important for oligomer formation may bepartially functional. The expression vector backbone is pVR1012x/s(VRC2000).

VRC5203

pVR1012x/s 89.6 Pgp160/h

The protein sequence of the envelope polyprotein (gp160) from 89.6P(Dual-tropic, GenBank accession number U89134/LOCUS: SIU89134) was usedto create a synthetic version of the gene (dual tropic gp160/h) usingcodons optimized for expression in human cells. The nucleotide sequence89.6P gp160/h shows little homology to the 89.6P gene, but the proteinencoded is the same. Full length SU and TM proteins are expressed fromthe pVR1012x/s (VRC2000) vector backbone

VRC5300

pVR1012x/s R5(clade C)gp140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from 92br025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (R5gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence R5gp160/h showslittle homology to the 92br025 gene, but the protein encoded is thesame. The full-length R5-tropic version of the envelope protein wassynthesized by Operon under the name: kongene. The XbaI (18 nt up-streamfrom ATG) to BglII (1376 nt down-stream from ATG) fragment whichcontains polylinker at the 5′ end, Kozak sequence and ATG was clonedinto the XbaI to BglII sites of VRC2701 pVR1012x/s X4gp140(del F/CL delH IS)/h backbone. Therefore, the gene is R5 (clade C) gp160/h up to theBglII site (1376 nt from ATG) and the rest of the gene after BglII siteis VRC2701 pVR1012x/s X4gp140(del F/CL del H IS)/h. The truncatedenvelope polyprotein contains the entire SU protein and a portion of theTM protein including the fusion domain, but lacking the transmembranedomain. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. The expressionvector backbone is pVR1012x/s (VRC2000). The full-length X4-tropicversion of the envelope protein from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 680.

VRC5301

pVR1012x/s R5(clade C)gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from 92br025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (R5gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence R5gp160/h showslittle homology to the 92br025 gene, but the protein encoded is thesame. The full-length R5-tropic version of the envelope protein wassynthesized by Operon under the name: kongene. The XbaI (18 nt up-streamfrom ATG) to BglII (1376 nt down-stream from ATG) fragment whichcontains polylinker at the 5′ end, Kozak sequence and ATG was clonedinto the XbaI to BglII sites of VRC2701 pVR1012x/s X4gp140(del F/CL delH IS)/h backbone. Therefore, the gene is R5 (clade C) gp160/h up to theBglII site (1376 nt from ATG) and the rest of the gene after BglII siteis VRC2707 pVR1012x/s X4gp145(del F/CL del H IS)/h. The truncatedenvelope polyprotein contains the entire SU protein and a portion of theTM protein including the fusion domain, but lacking the transmembranedomain. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 503-536, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 593-620, have been deleted. The expressionvector backbone is pVR1012x/s (VRC2000). The full-length X4-tropicversion of the envelope protein from pX4gp160/h (VRC3300) was terminatedafter the codon for amino acid 704.

VRC5303

pVR1012x/s R5gp145CladeC(Brazil) delCFI/h

Authentic Clade C unlike 5301 which is a hybrid between C and B.

VRC 5304

pVR1012x/s R5(clade A)gp 140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from 92rw020(R5-tropic, GenBank accession number U51283) was used to create asynthetic version of the gene (Clade-A gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the 92rw020 gene, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1837nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein contains theentire SU protein and the TM domain, but lacking the Fusion domain andCytoplasmic domain. Regions important for oligomer formation may bepartially functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 486-519, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 576-604, have been deleted. Theexpression vector backbone is pVR1102x/s (VRC2000).

VRC 5305

pVR1012x/s R5(clade A)gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp160) from 92rw020(R5-tropic, GenBank accession number U51283) was used to create asynthetic version of the gene (Clade-A gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the 92rw020 gene, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1912nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein contains theentire SU protein and the TM domain, but lacking the Fusion domain andCytoplasmic domain. Regions important for oligomer formation may bepartially functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 486-519, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 576-604, have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5306

pVR1012x/s R5(clade E)gp140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp140delCFI) from93th966.8 (R5-tropic, GenBank accession number U08456) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 93th966.8, but theprotein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1856 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains the entire SU protein and the TM domain, but lacking the Fusiondomain and Cytoplasmic domain. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 497-530, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 588-613,have been deleted. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 5307

pVR1012x/s R5(clade E)gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp145delCFI) from93th966.8 (R5-tropic, GenBank accession number U08456) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 93th966.8, but theprotein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1928 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains the entire SU protein and the TM domain, but lacking the Fusiondomain and Cytoplasmic domain. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 497-530, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 588-613,have been deleted. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 5308

pVR1012x/s R5(clade C South African)gp140(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1833nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein contains theentire SU protein and the TM domain, but lacking the Fusion domain andCytoplasmic domain. Regions important for oligomer formation may bepartially functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 487-520, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 577-605, have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5309

pVR1012x/s R5(clade C South African)gp145(del F/CL del H IS)/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein contains theentire SU protein and the TM domain, but lacking the Fusion domain andCytoplasmic domain. Regions important for oligomer formation may bepartially functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) arerequired for oligomerization. The Fusion and Cleavage (F/CL) domains,from amino acids 487-520, have been deleted. The Interspace (IS) betweenHeptad (H) 1 and 2, from amino acids 577-605, have been deleted. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5350

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV1

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1 loop(a.a.133-148) of the SU protein. It alsolacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembranedomain, and cytoplasmic domain in the TM protein. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5351

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV12

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2 loops (a.a.133-191) of the SU protein.It also lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembranedomain, and cytoplasmic domain in the TM protein. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5352

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV123

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V3 loops (a.a.130-191, 330-358) of theSU protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5353

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV1234

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V3, V4 loops (a.a.130-191, 330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), transmembrane domain, and cytoplasmic domain in the TMprotein. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 5354

pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV124

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V4 loops (a.a. 130-191, 384-408) of theSU protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5355

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV13

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V3 loops (a.a.133-148, 330-358) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5356

pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV134

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V3, V4 loops (a.a.130-148, 330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), transmembrane domain, and cytoplasmic domain in the TMprotein. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 5357

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV14

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V4 loops (a.a. 130-148, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5358

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV2

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2 loop(a.a.154-191) of the SU protein. It alsolacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembranedomain, and cytoplasmic domain in the TM protein. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5359

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV23

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140 (delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V3 loops (a.a. 154-191, 330-358) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5360

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV234

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V3, V4 loops (a.a.154-191, 330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), transmembrane domain, and cytoplasmic domain in the TMprotein. Regions important for oligomer formation may be partiallyfunctional: Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC 5361

pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV24

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V4 loops (a.a. 154-191, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5362

pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV3

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V3 loop(a.a.330-358) of the SU protein. It alsolacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembranedomain, and cytoplasmic domain in the TM protein. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5363

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV34

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V3, V4 loops (a.a.330-358, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),transmembrane domain, and cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5364

pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV4

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp140delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp140(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V4 loop(a.a.384-408) of the SU protein. It alsolacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembranedomain, and cytoplasmic domain in the TM protein. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5365

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV1

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1 loop(a.a.133-148) of the SU protein. It alsolacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), andCytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5366

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV12

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2 loops (a.a.133-191) of the SU protein.It also lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), andCytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5367

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV123

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V3 loops (a.a. 133-191, 330-358) of theSU protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5368

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV1234

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V3, V4 loops (a.a. 133-191,330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), and Cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5369

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV124

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V2, V4 loops (a.a. 133-148,154-191,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), and Cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5370

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV13

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V3 loops (a.a.133-148, 330-358) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5371

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV134

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V3, V4 loops (a.a. 133-148,330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), and Cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5372

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV14

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V1, V4 loops (a.a.133-148, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5373

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV2

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2 loop (a.a.154-191) of the SU protein. Italso lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), andCytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5374

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV23

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V3 loops (a.a.154-191, 330-358) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5375

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV234

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V3, V4 loops (a.a. 154-191,330-358,384-408) of the SU protein. It also lacks the Fusion and Cleavage (F/CL)domains(a.a. 496-529), the Interspace (IS) between Heptad (H) 1 and2(a.a. 586-612), and Cytoplasmic domain in the TM protein. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC 5376

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV24

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V2, V4 loops (a.a.154-191, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5377

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV3

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V3 loop (a.a.330-358) of the SU protein. Italso lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), andCytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5378

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV34

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V3, V4 loops (a.a.330-358, 384-408) of the SUprotein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),and Cytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC 5379

PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV4

The protein sequence of the envelope polyprotein (gp160) from 92BR025(R5-tropic, GenBank accession number U52953) was used to create asynthetic version of the gene (Brazil-C gp145delCFI) using codonsoptimized for expression in human cells. The nucleotide sequence ofBrazil-C gp145(delCFI) shows little homology to the gene 92BR025, butthe protein encoded is the same. The XbaI (18 nt up-stream from ATG) toBamH1 (1910 nt down-stream from ATG) fragment which contains polylinkerat the 5′ end, Kozak sequence and ATG was cloned into the XbaI to BamH1sites of pVR1012x/s backbone. The truncated envelope polyproteincontains deletion in the V4 loop (a.a.384-408) of the SU protein. Italso lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), theInterspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), andCytoplasmic domain in the TM protein. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5500

pVR1012x/s R5(SA-C)gp 140(dCFI) dV1/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1loop (a.a.136-150), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),the transmembrane domain and the intracellular region. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5501

pVR1012x/s R5(SA-C)gp140(dCFI) dV12/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V2loops (a.a.136-194), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),the transmembrane domain and the intracellular region. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC 5502

pVR1012x/s R5(SA-C)gp140(dCFI) dV23/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V3 loops (a.a. 136-194, 297-325), the Fusion and Cleavage (F/CL)domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5503

pVR1012x/s R5(SA-C)gp 140(dCFI)dV1234/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V3, V4 loops (a.a.136-194,297-325, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), the transmembrane domain and the intracellularregion. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC5504

pVR1012x/s R5(SA-C)gp 140(dCFI)dV124/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1102x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V4 loops (a.a. 136-194, 385-399), the Fusion and Cleavage (F/CL)domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC550S

pVR1012x/s R5(SA-C)gp 140(dCFI)dV13/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V3loops (a.a.136-150, 297-325), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5506

pVR1012x/s R5(SA-C)gp140(dCFI)dV134/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V3, V4 loops (a.a. 136-150, 297-325, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), the transmembrane domain and the intracellularregion. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC5507

pVR1012x/s R5(SA-C)gp140(dCFI)dV14/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V4loops (a.a.136-150, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5508

pVR1012x/s R5(SA-C)gp 140(dCFI)dV2/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2loop (a.a.156-194), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),the transmembrane domain and the intracellular region. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1102x/s (VRC2000).

VRC5509

pVR1012x/s R5(SA-C)gp140(dCFI)dV23/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2, V3loops (a.a.156-194, 297-325), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5510

pVR1012x/s R5(SA-C)gp 140(dCFI)dV234/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2,V3, V4 loops (a.a. 156-194, 297-325, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), the transmembrane domain and the intracellularregion. Regions important for oligomer formation may be partiallyfunctional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are requiredfor oligomerization. The expression vector backbone is pVR1012x/s(VRC2000).

VRC5511

pVR1012x/s R5(SA-C)gp140(dCFI)dV24/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2, V4loops (a.a.156-194, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5512

pVR1012x/s R5(SA-C)gp 140(dCFI)dV3/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V3loop (a.a.297-325), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),the transmembrane domain and the intracellular region. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5513

pVR1012x/s R5(SA-C)gp 140(dCFI)dV34/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V3, V4loops (a.a. 297-325, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), the transmembrane domain and the intracellular region.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The expression vector backbone is pVR1012x/s (VRC2000).

VRC5514

pVR1012x/s R5(SA-C)gp140(dCFI)dV4/h

The protein sequence of the envelope polyprotein (gp140delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp140delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V4loop (a.a 385-399), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),the transmembrane domain and the intracellular region. Regions importantfor oligomer formation may be partially functional. Heptad(H) 1, Heptad2 and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5515

pVR1012x/s R5(SA-C)gp 145(dCFI)dV1/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1loop (a.a.136-150), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),and the intracellular region. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5516

pVR1012x/s R5(SA-C)gp145(dCFI)dV12/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V2loops (a.a.136-194), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),and the intracellular region. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5517

pVR1012x/s R5(SA-C)gp 145(dCFI)dV123/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V3 loops (a.a.136-194, 297-325), the Fusion and Cleavage (F/CL)domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5518

pVR1012x/s R5(SA-C)gp 145(dCFI)dV1234/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V3, V4 loops (a.a.136-194, 297-325, 385-399), the Fusion andCleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) betweenHeptad (H) 1 and 2 (a.a.577-605), and the intracellular region. Regionsimportant for oligomer formation may be partially functional. Heptad(H)1, Heptad 2 and their Interspace(IS) are required for oligomerization.The expression vector backbone is pVR1012x/s (VRC2000).

VRC5519

pVR1012x/s R5(SA-C)gp 145(dCFI)dV2/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2loop (a.a.156-194), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),and the intracellular region. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5520

pVR1012x/s R5(SA-C)gp 145(dCFI)dV23/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2, V3loops (a.a.156-194, 297-325), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5521

pVR1012x/s R5(SA-C)gp145(dCFI)dV234/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2,V3, V4 loops (a.a.156-194, 297-325, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5522

pVR1012x/s R5(SA-C)gp 145(dCFI)dV24/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V2, V4loops (a.a.156-194, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5523

pVR1012x/s R5(SA-C)gp145(dCFI)dV3/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V3loop (a.a.297-325), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),and the intracellular region. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5524

pVR1012x/s R5(SA-C)gp145(dCFI)dV34/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V3, V4loops (a.a. 297-325, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5525

pVR1012x/s R5(SA-C)gp 145(dCFI)dV4/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V4loop (a.a.385-399), the Fusion and Cleavage (F/CL) domains (a.a.487-520), the Interspace (IS) between Heptad (H) 1 and 2 (a.a.577-605),and the intracellular region. Regions important for oligomer formationmay be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The expression vectorbackbone is pVR1012x/s (VRC2000).

VRC5526

pVR1012x/s R5(SA-C)gp 145(dCFI)dV13/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V3loops (a.a.136-150, 297-325), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5527

pVR1012x/s R5(SA-C)gp145(dCFI)dV134/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V3, V4 loops (a.a.136-150, 297-325, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5528

pVR1012x/s R5(SA-C)gp145(dCFI)dV124/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1,V2, V4 loops (a.a.136-150, 156-194, 385-399), the Fusion and Cleavage(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1and 2 (a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

VRC5529

pVR1012x/s R5(SA-C)gp 145(dCFI)dV14/h

The protein sequence of the envelope polyprotein (gp145delCFI) from97ZA012 (R5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp145delCFI) usingcodons optimized for expression in human cells. The nucleotide sequenceR5gp145delCFI shows little homology to the gene 97ZA012, but the proteinencoded is the same. The XbaI (18 nt up-stream from ATG) to BamH1 (1914nt down-stream from ATG) fragment which contains polylinker at the 5′end, Kozak sequence and ATG was cloned into the XbaI to BamH1 sites ofpVR1012x/s backbone. The truncated envelope polyprotein lacks the V1, V4loops (a.a.136-150, 385-399), the Fusion and Cleavage (F/CL) domains(a.a. 487-520), the interspace (IS) between Heptad (H) 1 and 2(a.a.577-605), and the intracellular region. Regions important foroligomer formation may be partially functional. Heptad(H) 1, Heptad 2and their Interspace(IS) are required for oligomerization. Theexpression vector backbone is pVR1012x/s (VRC2000).

Gag-Pol Plasmids VRC3900

pVR1012x/s Gag/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic gag gene was ligated in frame with sequences encoding thepol polyprotein to produce pGag(fs)Pol/h (VRC4200). The protein sequenceof the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBankaccession number M19921) was used to create a synthetic version of thepol gene using codons optimized for expression in human cells. Toproduce a gene that expresses all of the gag proteins, the regionencoding pol amino acids 77-1003 were deleted from Gag(fs)Pol/h toproduce Gag/h. This construct also encodes most of the protease (98amino acids) gene encoding amino acids 3-77. Gag/h is expressed from thepVR1012x/s vector backbone.

VRC3901

pVR1012x/s SIV Gag/h

The protein sequence of the gag polyprotein (amino acid from 1-550)SIVmac239 (GenBank accession number M33262) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the SIVMac239 gene, but the protein encoded is thesame.

VRC4000

pVR1012x/s Gag-Pol/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. Gag-Pol/h is expressed from the pVR1012x/s vector backbone.

VRC4001

pVR1012x/s SIVGag-Pol/h

Eukaryotic vector with humanized codons expressing the Gag-pol gene ofSIVmac239. The Sal 1-XbaI fragment of SIV gag(VRC3901) was inserted intoSal 1-XbaI of SIV Pol(VRC4101) to create VRC4001.

VRC4100

pVR1012x/s Pol/h

The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene using codons optimized for expression in humancells. To initiate translation at the beginning of Pol, a methioninecodon was added to the 5′-end of the synthetic polymerase gene to createthe Pol/h gene. Pol/h is expressed from the pVR1012x/s (VRC2000) vectorbackbone.

VRC4101

pVR1102x/s SIV Pol/h

The protein sequence of the pol polyprotein (amino acids 3-1017) fromSIVmac239 (GenBank accession number M19921) was used to create asynthetic version of the pol gene using codons optimized for expressionin human cells. To initiate translation at the beginning of Pol, amethionine codon was added to the 5′-end of the synthetic polymerasegene to create the Pol/h gene. The Protease (Pr) mutation is at polamino acid 123 and is AAG->GGA or amino acids R->G. Reversetranscriptase(RT) mutation is at aa 352(GAC to CAT D(D to H)-RT) andIntegrase mutation is at aa 788(GAC to GGC(D to A)-IN). Pol/h isexpressed from the pVR1012x/s (VRC2000) vector backbone.

VRC4200

pVR1012x/s Gag(fs)Pol/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. Gag(fs)Pol/h is expressed from the pVR1012x/svector backbone.

VRC4300

pVR1012 Gag-Pol(d delta RT delta IN)/h

Eukaryotic vector with humanized codons expressing the Gag and the frameshifted Pol genes of HIV HXB2 subtype B with deletions in Reversetranscriptase, and Integrase regions. The protein sequence of the gagpolyprotein (Pr55, amino acids 1-432) from HXB2 (GenBank accessionnumber K03455) was used to create a synthetic version of the gag geneusing codons optimized for expression in human cells. The nucleotidesequence of the synthetic gag gene shows little homology to the HXB2gene, but the protein encoded is the same. The synthetic gag genecontains all of the mature Gag proteins except for the last two that arenormally cleaved from the carboxy-terminus of the gag polyprotein, p1and p6 (amino acids 433-500). The synthetic gag gene was ligated inframe with sequences encoding the pol polyprotein. The protein sequenceof the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBankaccession number M19921) was used to create a synthetic version of thepol gene (Pol/h) using codons optimized for expression in human cells.To create the possibility for translational frameshifting as means toexpress the gag-pol polyprotein, the synthetic coding region for thelast four amino acids of the NC protein through the rest of gag plus anadditional 3 amino acids from pol were replaced with the correspondingviral sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3(GenBank accession number M19921). The substitution of viral forsynthetic sequences both introduces the sites required for frameshiftingand restores the ability to express all gag proteins, including p1 andp6 The Reverse Transcriptase (RT) mutation is at gag-pol amino acid 771and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation is atgag-pol amino acid 1209 and is ACT->CAT or amino acids-D->A. Note: Thisvector is not in the pVR1012x/s backbone.

VRC4301

pVR1012x/s-Gag(FS)-Pol-delta RT IN-IRES-R5gp157-Nef

For the Gag(FS)Pol delta RT delta IN/h portion, VRC4302 pVR1012x/sGag(fs)Pol(delta PR delta/h was used for the protein sequence of the gagpolyprotein (Pr55, amino acids 1-432) from HXB2 (GenBank accessionnumber K03455) was used to create a synthetic version of the gag geneusing codons optimized for expression in human cells. The nucleotidesequence of the synthetic gag gene shows little homology to the HXB2gene, but the protein encoded is the same. The synthetic gag genecontains all of the mature Gag proteins except for the last two that arenormally cleaved from the carboxy-terminus of the gag polyprotein, p1and p6 (amino acids 433-500). The synthetic gag gene was ligated inframe with sequences encoding the pol polyprotein. The protein sequenceof the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBankaccession number M19921) was used to create a synthetic version of thepol gene (Pol/h) using codons optimized for expression in human cells.To create the possibility for translational frameshifting as means toexpress the gag-pol polyprotein, the synthetic coding region for thelast four amino acids of the NC protein through the rest of gag plus anadditional 3 amino acids from pol were replaced with the correspondingviral sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3(GenBank accession number M19921). The substitution of viral forsynthetic sequences both introduces the sites required for frameshiftingand restores the ability to express all gag proteins, including p1 andp6. Gag(fs)Pol/h is expressed from the pVR1012x/s vector backbone. TheReverse Transcriptase (RT) mutation is at gag-pol amino acid 771 and isGAC->CAC or amino acids D->H. The Integrase (IN) mutation is at gag-polamino acid 1209 and is ACT->CAT or amino acids D->A. This gene has beenfused to an Internal Ribosomal Entry Site (IRES) and then fused to theR5gp157-Nef from VRC2200 pVR1012x/s R5gp157-NefDMHCDCD4/h in which theprotein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. The nucleotide sequence X4gp160/h showslittle homology to the HXB2 gene, but the protein encoded is the samewith the following amino acid substitutions: F53L, N94D, K192S, I215N,A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic versionof the envelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GeneBank accession number M68893, again using human preferred codons).The envelope-Nef fusion protein expressed from pR5gp157-Nef/h containsthe first 820 amino acids from the HIV envelope glycoprotein (gp 157)fused to the entire mutant Nef protein. The gene for gp157 was ligatedin frame with the full-length mutant Nef gene from pNefDMHCDCD4/h(VRC3600) to produce pR5gp157-NefDMHCDCD4/h. The protein sequence of theNef protein from HIV-1 PV22 (GenBank accession number K02083) was usedto create a synthetic version of the Nef gene (Nef/h) using codonsoptimized for expression in human cells. To disrupt the ability of Nefto limit both MHC class I and CD4 expression, point mutations wereintroduced into the Nef gene from pNef/h (VRC3500). The resulting aminoacids substitutions in pNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174Aand D175A. R5gp157-NefDMHCDCD4/h is expressed from the pVR1012x/s(VRC2000) vector backbone.

VRC4302

pVR1012 Gag(delFS)Pol(delta PR delta RT delta IN)/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. Note:This vector is not in the pVR1012x/s backbone.

VRC4303

pVR1012SIV Gag(delFS)Pol(delta PR delta RT delta IN)/h

Eukaryotic vector with humanized codons expressing the Gag and theframe-shift-deleted Pol genes SIVmac239 with deletions in the Protease,Reverse transcriptase, and Integrase regions. The 5 of Ts from 3188 to3192 of SIV gag-pol(VRC4001) were deleted to create VRC4303.

VRC4304

pVR1012 Gag (delFS)Pol delta PR delta RT delta IN/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HIV-1 C clade(GenBank accession number U52953) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HIV-1 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. Note:This vector is not in the pVR1012x/s backbone.

VRC4305

pVR1012 Gag-A(delFS)Pol(delta PR delta RT delta IN)/h

Eukaryotic vector with humanized-codons expressing the Gag and the frameshifted Pol genes of HIV HIV-1A clade with deletions in the Protease,Reverse transcriptase, and Integrase regions. VRC4305 pVR1012Gag(-AdelFS)Pol(delta PR delta RT delta IN)/h The protein sequence ofthe gag polyprotein (Pr55, amino acids 1-432) from HIV-1 A clade(GenBank accession number AF004885) was used to create a syntheticversion of the gag gene using codons optimized for expression in humancells. The nucleotide sequence of the synthetic gag gene shows littlehomology to the HIV-1 gene, but the protein encoded is the same. Thesynthetic gag gene contains all of the mature Gag proteins except forthe last two that are normally cleaved from the carboxy-terminus of thegag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gag genewas ligated in frame with sequences encoding the pol polyprotein. Theprotein sequence of the pol polyprotein (amino acids 3-1003) from NL4-3(GenBank accession number M19921) was used to create a synthetic versionof the pol gene (Pol/h) using codons optimized for expression in humancells. To create the possibility for translational frameshifting asmeans to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. Note:This vector is not in the pVR1012x/s backbone.

pVRC4306pVR1012 Gag(delFS)Pol delta PR delta RT delta IN/Nef/h

The protein sequence of the Gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the Gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic Gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic Gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe Gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic Gaggene was ligated in frame with sequences encoding the Pol polyprotein.The protein sequence of the Pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the Pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the Gag-Pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of Gagplus an additional 3 amino acids from Pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all Gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. The Protease (PR)mutation is at Gag-Pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at Gag-Pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat Gag-Pol amino acid 1209 and is ACT->CAT or amino acids D->A. TheNef/h gene was fused to downstream of Pol gene of Gag(delFS)Pol(delta PRdelta RT delta IN). No loss or extra-amino acid was created by thefusion between Nef and Pol. The ATG of Nef was preserved. The proteinsequence of the Nef protein from HIV-1 PV22 (GenBank accession numberK02083) was used to create a synthetic version of the Nef gene (Nef/h)using codons optimized for expression in human cells. The nucleotidesequence Nef/h shows little homology to the viral gene, but the proteinencoded is the same. Note: This vector is not in the pVR1012x/sbackbone.

VRC 4308 VRC4302-myr

pVR1012 Gag (delFS) Pol deltaPR deltaRT deltaIN deltaMyr/h

The myristylation site was deleted from pVRC4302. The protein sequenceof the gag polyprotein (Pr55, amino acids 1-432) from HXB2 (GenBankaccession number K03455) was used to create a synthetic version of thegag gene using codons optimized for expression in human cells. Thenucleotide sequence of the synthetic gag gene shows little homology tothe HXB2 gene, but the protein encoded is the same. The synthetic gaggene contains all of the mature Gag proteins except for the last twothat are normally cleaved from the carboxy-terminus of the gagpolyprotein, p1 and p6 (amino acids 433-500). The synthetic gag gene wasligated in frame with sequences encoding the pol polyprotein. Theprotein sequence of the pol polyprotein (amino acids 3-1003) from NL4-3(GenBank accession number M19921) was used to create a synthetic versionof the pol gene (Pol/h) using codons optimized for expression in humancells. To create the possibility for translational frameshifting asmeans to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. Themyristylation site was deleted. Note: This vector is not in thepVR1012x/s backbone.

VRC 4309

pVR1012 Gag (delFS) Pol deltaPR deltaRT deltaIN delta Myr/Nef/h

The myristylation site was deleted from pVRC4306. The protein sequenceof the gag polyprotein (Pr55, amino acids 1-432) from HXB2 (GenBankaccession number K03455) was used to create a synthetic version of thegag gene using codons optimized for expression in human cells. Thenucleotide sequence of the synthetic gag gene shows little homology tothe HXB2 gene, but the protein encoded is the same. The synthetic gaggene contains all of the mature Gag proteins except for the last twothat are normally cleaved from the carboxy-terminus of the gagpolyprotein, p1 and p6 (amino acids 433-500). The synthetic gag gene wasligated in frame with sequences encoding the pol polyprotein. Theprotein sequence of the pol polyprotein (amino acids 3-1003) from NL4-3(GenBank accession number M19921) was used to create a synthetic versionof the pol gene (Pol/h) using codons optimized for expression in humancells. To create the possibility for translational frameshifting asmeans to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. The stopcodon TAG was removed and synthetic B clade Nef (Genbank access number)was fusion to the 3′ end of pol by PCR. Note: This vector is not in thepVR1012x/s backbone.

VRC 4310

pVR1012Nef Gag (del fs) (del Myr) Pol (delta PR delta RT delta IN)/h

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HXB2 (GenBank accession number K03455) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HXB2 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. The stopcodon TAG of synthetic B clade Nef (Genbank access number) gene wasremoved and was fused to the 5′ end of gene by PCR. Note: This vector isnot in the pVR1012x/s backbone.

VRC 4311

pVR1012 Gag-C(delFS)Pol(delta PR delta RT delta IN) Nef/h

VRC4304+Clade C Nef

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HIV-1 C clade(GenBank accession number U52953) was used to create asynthetic version of the gag gene using codons optimized for expressionin human cells. The nucleotide sequence of the synthetic gag gene showslittle homology to the HIV-1 gene, but the protein encoded is the same.The synthetic gag gene contains all of the mature Gag proteins exceptfor the last two that are normally cleaved from the carboxy-terminus ofthe gag polyprotein, p1 and p6 (amino acids 433-500). The synthetic gaggene was ligated in frame with sequences encoding the pol polyprotein.The protein sequence of the pol polyprotein (amino acids 3-1003) fromNL4-3 (GenBank accession number M19921) was used to create a syntheticversion of the pol gene (Pol/h) using codons optimized for expression inhuman cells. To create the possibility for translational frameshiftingas means to express the gag-pol polyprotein, the synthetic coding regionfor the last four amino acids of the NC protein through the rest of gagplus an additional 3 amino acids from pol were replaced with thecorresponding viral sequences (nucleotides 2074-2302 on the HXB2 genome)from NL4-3 (GenBank accession number M19921). The substitution of viralfor synthetic sequences both introduces the sites required forframeshifting and restores the ability to express all gag proteins,including p1 and p6. The deleted Frame Shifted (delFS) has 5 Tnucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h isexpressed from the pVR1012x/s vector backbone. The Protease (PR)mutation is at gag-pol amino acid 553 and is AGG->GGC or amino acidsR->G. The Reverse Transcriptase (RT) mutation is at gag-pol amino acid771 and is GAC->CAC or amino acids D->H. The Integrase (IN) mutation isat gag-pol amino acid 1209 and is ACT->CAT or amino acids D->A. The stopcodon TAG was removed and synthetic C clade Nef (Genbank accessionnumber: U52953) was fusion to the 3′ end of pol by PCR. Note: Thisvector is not in the pVR1012x/s backbone.

VRC 4312 Gag (del fs) (del Myr) Nef Pol ΔPRΔRTΔIN/hVRC-Myr-gag-Dnef-Dpol

(Eukaryotic Vector with Humanized Codons Expressing the Gag, TruncatedNef, and Truncated Pol Proteins of HIV Subtype B.Ns.)

This construct was derived from VRC4302. The protein sequence of the gagpolyprotein (Pr55, amino acids 1-432) from HXB2 (GenBank accessionnumber K03455) was used to create a synthetic version of the gag geneusing codons optimized for expression in human cells. The nucleotidesequence of the synthetic gag gene shows little homology to the HXB2gene, but the protein encoded is the same. The synthetic gag genecontains all of the mature Gag proteins except for the last two that arenormally cleaved from the carboxy-terminus of the gag polyprotein, p1and p6 (amino acids 433-500). The synthetic gag gene was ligated inframe with sequences encoding synthetic nef gene that 51 aa were deletedfrom 5′. 77 aa were deleted from 5′ of pol polyprotein, and ligated with3′ of nef in which tag stop codon was deleted. The protein sequence ofthe pol polyprotein (amino acids 3-78-1003) from NL4-3 (GenBankaccession number M19921) was used to create a synthetic version of thepol gene (Pol/h) using codons optimized for expression in human cells.To create the possibility for translational frameshifting as means toexpress the gag-pol polyprotein, the synthetic coding region for thelast four amino acids of the NC protein through the rest of gag plus anadditional 3 amino acids from pol were replaced with the correspondingviral sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3(GenBank accession number M19921). The substitution of viral forsynthetic sequences both introduces the sites required for frameshiftingand restores the ability to express all gag proteins, including p1 andp6. The deleted Frame Shifted (delFS) has 5 T nucleotides deletedbetween the Gag and Pol sequences. Gag(fs)Pol/h is expressed from thepVR1102x/s vector backbone. The Protease (PR) mutation is at gag-polamino acid 553 and is AGG->GGC or amino acids R->G. The ReverseTranscriptase (RT) mutation is at gag-pol amino acid 771 and is GAC->CACor amino acids D->H. The Integrase (IN) mutation is at gag-pol aminoacid 1209 and is ACT->CAT or amino acids D->A. Note: This vector is notin the pVR012x/s backbone.

VRC 4313

pVR1012 Gag Clade A (del fs)Pol(Δ PR Δ RT Δ IN)/h

VRC4305+Clade A Nef

The protein sequence of the gag polyprotein (Pr55, amino acids 1-432)from HIV-1 A clade (GenBank accession number AF004885) was used tocreate a synthetic version of the gag gene using codons optimized forexpression in human cells. The nucleotide sequence of the synthetic gaggene shows little homology to the HIV-1 gene, but the protein encoded isthe same. The synthetic gag gene contains all of the mature Gag proteinsexcept for the last two that are normally cleaved from thecarboxy-terminus of the gag polyprotein, p1 and p6 (amino acids433-500). The synthetic gag gene was ligated in frame with sequencesencoding the pol polyprotein. The protein sequence of the polpolyprotein (amino acids 3-1003) from NL4-3 (GenBank accession numberM19921) was used to create a synthetic version of the pol gene (Pol/h)using codons optimized for expression in human cells. To create thepossibility for translational frameshifting as means to express thegag-pol polyprotein, the synthetic coding region for the last four aminoacids of the NC protein through the rest of gag plus an additional 3amino acids from pol were replaced with the corresponding viralsequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3 (GenBankaccession number M19921). The substitution of viral for syntheticsequences both introduces the sites required for frameshifting andrestores the ability to express all gag proteins, including p1 and p6.The deleted Frame Shifted (delFS) has 5 T nucleotides deleted betweenthe Gag and Pol sequences. Gag(fs)Pol/h is expressed from the pVR1012x/svector backbone. The Protease (PR) mutation is at gag-pol amino acid 553and is AGG->GGC or amino acids R->G. The Reverse Transcriptase (RT)mutation is at gag-pol amino acid 771 and is GAC->CAC or amino acidsD->H. The Integrase (IN) mutation is at gag-pol amino acid

1209 and is ACT->CAT or amino acids D->A. The stop codon TAG was removedand synthetic A clade Nef (Genbank accession number:AF069670) was fusedto the 3′ end of pol by PCR. Note: This vector is not in the pVR1012x/sbackbone.

EXAMPLES Env Immunogens

Plasmids expressing the CXCR4-tropic HIV-1 HXB2 Env were madesynthetically with sequences designed to disrupt viral RNA structuresthat limit protein expression by using codons typically found in humancells. Briefly, the synthetic Env gene of HXB2 (GenBank accession numberK03455) was generated in three fragments by assembling the overlappingsynthetic oligonucleotides using PCR amplification. To produce aCCR5-tropic version of the HIV-1 envelope, the region encoding aminoacids 275 to 361 of HXB2 (CXCR4-tropic) gp160 was replaced withCCR5-tropic HIV-1 BaL sequence (GenBank accession number M68893), whichincludes the V3 loop. Glycosylation mutants were generated bysite-directed mutagenesis to replace asparagine with glutamic acidresidues at seventeen conserved glycosylation sites between amino acids88 and 448. To express truncation mutant Env proteins, stop codons wereintroduced after positions 752, 704, 680 or 592 to produce gp150, gp145,gp140, or gp128, respectively. The Env protein was further changed bydeleting amino acids 503 to 537 and 593 to 619, which removes thecleavage site sequence, the fusion domain, and a part of the spacerbetween the two heptad repeats. The structures of the synthetic HIVenvelope genes are shown (FIG. 178). The cDNAs were cloned in theexpression vector pNGVL or pVR1012 under the control of thecytomegalovirus immediate-early enhancer, promoter, and first intron.The protein sequence was identical to HXB2 Env except for the followingamino acid substitutions: F53L, N94D, K192S, I215N, A224T, A346D, P470L,T7231, and S745T.

Expression of Envelope Proteins in Transfected Cells

293 cells (10⁶) were plated in 60 mm dishes. Cells were transfected onthe following day with 2 μg of plasmid using calcium phosphate. Cellswere harvested 48 hours after transfection and lysed in buffercontaining 50 mM HEPES pH 7.0, 250 mM NaCl and 0.5% NP40. The proteinconcentration in the lysates was determined using the Bradford reagent(Bio-Rad). Proteins (25 μg) in lysates were separated by 7.5% SDS-PAGEand transferred to Immobilon-P membrane (Millipore, Bedford, Mass.). Envwas detected by immunoprecipitation followed by Western blotting usingpolyclonal antibody against gp160 (Intracel, Rockville, Md.).

Cell Surface Expression of Envelope by FACS Analysis

293 cells were harvested 48 hours after transfection and washed twicewith phosphate-buffered saline (PBS) containing 1% bovine serum albuminand incubated for 30 minutes on ice with polyclonal immunoglobulin froman HIV-1 infected patient. The secondary antibody against human IgGconjugated with FITC (Jackson Immuno Research) was added, incubated for30 minutes on ice, washed 3 times with PBS, and analyzed by flowcytometry (FACScan). The median fluorescent intensity values werederived using Cell Quant software.

DNA Injection in Mice

Six week old, female BALB/c mice were injected intramuscularly with 100μg of purified plasmid DNA suspended in 200 μl of normal saline. Foreach plasmid DNA, a group of 4 mice was injected three times atintervals of two weeks. The mice were bled two weeks after the lastinjection, sera collected and stored at 4° C.

Quantitation of the Antibody Response

Immunoprecipitation and Western blotting was used to detect theantibodies that bind to native envelope proteins. Sera from immunizedmice were used to immunoprecipitate gp160 from cell lysates ofgp160-transfected 293 cells. Indicated dilutions were used toimmunoprecipitate gp160 from the cell lysate (400 μg). Immunocomplexeswere separated by 7.5% SDS-PAGE and analyzed by immunoblotting using thepolyclonal antibody against gp 160.

Analysis of CTL Response

Spleens were removed aseptically and gently homogenized to a single cellsuspension, washed, and resuspended to a final concentration of 5×10⁷cells/ml. Cells were incubated for 7 days in presence of IL-2 (10 U/ml)and either irradiated peptide-pulsed splenocytes from naive mice or anirradiated stable cell line expressing the full length gp160 BC-env/rev.Three types of target cells were used: peptide-pulsed P815 cells (ATCCTIB64), BC10ME cells stably expressing gp160, and BC10ME cells pulsedwith peptides derived from gp160 sequence. Target cells were labeledwith ⁵¹Cr for 90 minutes and washed three times with RPMI-1640 with 10%FBS, 2 mM glutamine, 5×10⁻ ⁵M β-mercaptoethanol and fungisone,250units/ml, and resuspended in this media. Cytolytic activity wasdetermined in triplicate samples using all different target celldilutions in a 5-hour ⁵¹Cr-release assay.

Gag and Pol Development of Synthetic HIV-1 Gag-Pol Expression Vectors

The protein sequences of Gag (amino acids 1-432) from HXB2 (GenBankaccession number K03455) and Pol (amino acids 3-1003) from NL4-3(GenBank accession number M19921) were reverse translated by the GCGPackage (Genetic Computer Group, Inc., Madison, Wis.) using codonsexpected for human cells. A 226-bp fragment spanning the frame shiftsite and the overlapping region of the two reading frames from NL4-3were retained to allow expression of Gag and Gag-Pol precursorpolyproteins in the same construct. 86 oligonucleotides covering 4325DNA base pairs with 5′ SalI and 3′ EcoRI sites were purchased from GIBCOLife Technologies. Each of the oligonucleotides was 75 base-pairs with25 nt of overlap. The codon optimized Gag-Pol gene (hGag-Pol) wasassembled by PCR with Pwo (Boehringer Mannheim) and Turbo Pfu(Stratagene) high fidelity DNA polymerase. The PCR conditions wereoptimized with a PCR optimization kit (Stratagene) on a gradientRobocycler (Stratagene). Full-length synthetic Gag-Pol gene was clonedinto the Sal I and blunted Bgl II site of the mammalian expressionvector pNGVL-3 (Xu, Ling et al., 1998, Nature Medicine, 4:37-42) andconfirmed by DNA sequencing. Three additional constructs were derivedfrom the hGag-Pol gene. 5 Thymidines (Ts) in the frame shift site (FS)of the hGag-Pol gene were deleted (ΔFS) and the protease was inactivatedby replacing AGG in protease to GGC(R42G) to create hGag-PolΔFSΔPr. 432amino acids of the NH2-terminal of hGag-Pol gene were deleted and an ATGstart codon was added to create the hPol gene. 925 amino acids of theCOOH-terminal hGag-Pol were deleted to create the hGag gene.hGagPolΔFSΔPr, hPol and hGag genes were expressed in the pNGVL-3plasmid, derived by insertion of a polylinker into pVR1012. The plasmidexpressing viral Gag-Pol, pCMVΔ8.2 was a kind gift.

Transient Transfection and Analysis of Expression

293T cells were maintained in Dulbecco's modified Eagle medium (DMEM;GIBCO-BRL), supplemented with 10% fetal bovine serum (FBS). Plasmid DNAswere purified with double cesium chloride sedimentation gradients.Approximately 3×10⁶ 293T cells were placed in a 10-cm dish one daybefore transfection. 10 μg of pCMVdR8.2 plasmid (containing the viralgag-pol gene), or 5 pg of pVR1012s (containing the codon altered genes),were used to transfect 293T cells, using the calcium phosphate method.Three days after transfection, cell lysates were prepared with RIPAbuffer (Boehringer Mannheim, Indianapolis, Ind.) and separated by 4-15%gradient SDS-polyacrylamide gel electrophoresis (PAGE), then transferredonto an Immobilon P membrane (Millipore). Membranes were then incubatedwith anti HIV-1-IgG (AIDS Research and Reference Reagent Program),monoclonal anti-p24 (ICN), or rabbit anti-RT (Intracel, Rockville, Md.).Bands were visualized using the ECL Western blotting detection reagent(Amersham Pharmacia Biotech, Piscataway, N.J.), as described by themanufacturer. Expression levels were determined using a phosphorimager.

Generation of Stably Transfected Cell Lines

hGag and hPol genes were individually subcloned into the Xho I and EcoRIsites of a retroviral vector, pPGS-CITE-Neo. Three plasmid systems wereused to produce recombinant retroviruses containing the hGag or hPolgenes. 48 hours after transfection, the supernatants were collected totransduce CT26 and BC10ME which are syngeneic to Balb/C mice, andselected in 0.8 mg/ml of G418 two days after infection. The positiveclones were screened and confirmed by Western blotting and maintained in10% FCS supplemented RPMI (GIBCO-BRL) with 0.5 mg/ml G418.

DNA Vaccination of Mice

Female BALB/C mice, 6-8 weeks old, were used for immunogenicity studies.For DNA vaccination, mice were immunized with 100 μl (0.5 μg/ml DNA and0.9% NaCl) in the quadriceps muscle of each hind leg every two weeks fora total of 4 injections.

CTL Assay

Animals were sacrificed after the last immunization and spleens removedfrom both naïve and immunized mice using aseptic techniques. Spleniclymphocytes were harvested and the chromium release CTL assay wasperformed in triplicate as previously described. The peptides used forsensitizing cells are as follows: Two peptide mixtures from the Gagprotein P17(88-115) and p24(62-76). Seven peptide mixtures from the Polprotein: 1) P66(175-189), 2) P66(179-193), 3) P66(183-197), 4)P66(187-201), 5) P66(223-237), 6) P66(227-241), 7) P66(367-381).

Measurement of Antibody Responses

Anti-p24 ELISA assays

The anti-p24 ELISA assay was performed in Immunlon ninety-six-wellplates (Dynet Technologies Inc., Chantilly, Va.). The plates were coatedwith 50 μl of purified recombinant HIV-11IIB p24 antigen (Intracel) at aconcentration of 2 μg/ml in PBS buffer, pH 7.4 (GIBCO) with 0.05% sodiumazide. Plates were washed 3× in PBS containing 3% BSA and 0.05% Tween20(blocking buffer) and incubated for 2 hours. Mouse sera were seriallydiluted from 1:100 to 1:12,800 in blocking buffer, added to thep24-coated plates and incubated overnight at 4° C. Plates were thenwashed four times with PBS (0.05% Tween 20), and incubated with goatanti-mouse IgG (1:10,000 dilution, Sigma) for 2 hours at roomtemperature. Plates were washed four times, and then pNPP alkalinephosphatase substrate (75 μl; Sigma, St. Louis, Mo.) was added to eachwell. The reaction was stopped after one hour by addition of 0.5 N NaOH(25 μl). The plates were read on an ELISA reader at 405 nm, and titerswere calculated at a cutoff optical density of 0.4.

HIV-1 immunoblotting

The strips containing HIV-1 proteins (Immunectics Inc., Cambridge,Mass.) were incubated with pooled mouse sera at a dilution of 1:25.Purified human anti-HIV IgG (AIDS Research and Reference ReagentProgram, Rockville, Md.) was used as a positive control. Bands werevisualized using the ECL Western blotting detection reagent (AmershamPharmacia Biotech, Piscataway, N.J.).

Immunoprecipitation and Western Blotting

Three days after transfection, hPol gene-transfected 293T cell lysateswere prepared with RIPA buffer. The pooled mouse sera were diluted withimmunoprecipitation (IP) buffer (100 mM KCl, 2.5 mM MgCl₂, 20 mM HEPES,pH 7.9, 0.1% NP-40, 1 mM DTT and proteinase inhibitors). After adding 10Hg of the cell lysate containing the HIV-1 Pol protein, the reactionswere incubated overnight on a rotator at 4° C. 250 μl of Protein G and ASepharose beads (10% V/V in IP buffer) were then added, and thereactions were incubated for 2 hours on a rotator at 4° C. The beadswere washed four times with IP buffer, resuspended in 30 μl of 1× samplebuffer and loaded onto SDS-PAGE. After transfer onto an Immobilon Pmembrane (Millipore), membranes were incubated with anti HIV-1-IgG (AIDSResearch and Reference Reagent Program). Bands were visualized using theECL Western blotting detection reagent (Amersham Pharmacia Biotech).

Prime/Boost Vaccination Strategy

Groups of guinea pigs (4/group) were immunized (primed) with the vectorslisted below, 3 times at 2 week intervals. Two weeks after the third DNAimmunization, blood was collected for immune analysis. 2-3 days afterthe blood collection, the animals received a boost with a correspondingadenoviral construct, AdApt (Sullivan, N. et al., 2000, Nature,408:605-608). Blood was again collected 2 weeks after the adenoviralboost. Blood was tested for neutralizing antibodies and (using an ELISA)the presence of antibody against the envelope. The groups are summarizedbelow.

Prime Adenoviral Boost gp140delCFI (R5) Adv gp140delCFI (R5) gp145delCFI(R5) Adv gp145delCFI (R5) gp145delCFI (R5) Adv gp140delCFI (R5)gp140delCFI (89.6P) Adv gp140delCFI (89.6P) gp145delCFI (89.6P) Advgp145delCFI (89.6P) gp145delCFI (89.6P) Adv gp140delCFI (89.6P) Advgp140delCFI (R5) Adv gp140delCFI (R5) Adv gp145delCFI (R5) Advgp145delCFI (R5) Adv gp145delCFI (R5) Adv gp140delCFI (R5) Advgp140delCFI (89.6P) Adv gp140delCFI (89.6P) Adv gp145delCFI (89.6P) Advgp145delCFI (89.6P) Adv gp145delCFI (89.6P) Adv gp140delCFI (89.6P)

Results

The level of antibody in the sera after adenoviral boost was 8- to10-fold higher than the level of antibody present after 3 injections ofDNA alone, in almost all of the cases. Neutralizing antibodies weredetected in the sera of animals immunized with either gp145delCFI DNA orgp140delCFI DNA followed by Advgp140delCFI.

Neutralization was also observed in animals immunized with gp145delCFIDNA followed by Adv gp145delCFI, and in animals both primed and boostedwith adenovirus.

Optimal neutralization was seen when animals were primed withgp145delCFI DNA followed by adenovirus expressing gp140delCFI in bothlade B (R5-Bal strain) and 89.6P (Clade B, dual tropic strain).

All neutralizing activity was able to be blocked with the V3 peptide ofthe corresponding strains.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications and publications referredto above are hereby incorporated by reference.

1. A nucleic acid encoding a modified HIV Env glycoprotein wherein saidmodified HIV Env glycoprotein has a termination codon at position 704.2. The nucleic acid of claim 1, wherein said Env is from an R5 tropicHIV strain.
 3. The nucleic acid of claim 1, wherein said Env is from anX4 tropic HIV strain.
 4. The nucleic acid of claim 1, wherein the codonsin said nucleic acid have been optimized for expression in humans. 5.The nucleic acid of claim 1, wherein at least one variable region hasbeen deleted.
 6. The nucleic acid of claim 5, wherein said at least onevariable region is the V1 loop.
 7. The nucleic acid of claim 6, whereinthe V2 loop has been deleted.
 8. The nucleic acid of claim 5, whereinsaid at least one variable region is selected from the group consistingof the V1 loop, the V2 loop, the V3 loop and the V4 loop.
 9. The nucleicacid of claim 1, wherein said modified HIV Env glycoprotein is from alade selected from the group consisting of Clade A, Clade B and Clade C.10. The nucleic acid of claim 9, wherein said modified HIV Envglycoprotein is from Clade C (South African).
 11. The nucleic acid ofclaim 1, wherein said nucleic acid encodes a modified HIV Envglycoprotein in which the cleavage site, fusion domain, and at leastpart of the interspace between the two heptad-repeats are deleted. 12.The nucleic acid of claim 1, wherein said nucleic acid comprises theinsert in SEQ ID NO: 92 which encodes a modified HIV Env glycoprotein.13. The nucleic acid of claim 1, wherein said nucleic acid comprises anucleotide sequence at least 95% identical to the nucleotide sequence ofthe insert in SEQ ID NO: 92 which encodes a modified HIV Envglycoprotein.
 14. The nucleic acid of claim 1, wherein said nucleic acidencodes a polypeptide at least 95% identical to the polypeptide encodedby the insert in SEQ ID NO: 92 which encodes a modified HIV Envglycoprotein.
 15. The nucleic acid of claim 1, wherein said nucleic acidcomprises the insert in SEQ ID NO: 14 which encodes a modified HIV Envglycoprotein.
 16. The nucleic acid of claim 1, wherein said nucleic acidcomprises a nucleotide sequence at least 95% identical to the nucleotidesequence of the insert in SEQ ID NO: 14 which encodes a modified HIV Envglycoprotein.
 17. The nucleic acid of claim 1, wherein said nucleic acidencodes a polypeptide at least 95% identical to the polypeptide encodedby the insert in SEQ ID NO: 14 which encodes a modified HIV Envglycoprotein.
 18. The nucleic acid of claim 1, wherein said nucleic acidcomprises the insert in SEQ ID NO: 96 which encodes a modified HIV Envglycoprotein.
 19. The nucleic acid of claim 1, wherein said nucleic acidcomprises a nucleotide sequence at least 95% identical to the nucleotidesequence of the insert in SEQ ID NO: 96 which encodes a modified HIV Envglycoprotein.
 20. The nucleic acid of claim 1, wherein said nucleic acidcomprises a nucleotide sequence at least 95% identical to the nucleotidesequence of the insert in SEQ ID NO: 96 which encodes a modified HIV Envglycoprotein.
 21. The nucleic acid of claim 1, wherein said nucleic acidcomprises the insert which encodes a modified HIV Env glycoprotein in asequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 954, SEQ ID NO: 55, SEQ ID NO: 56, SEQID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61,SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 86, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 142, SEQ ID NO: SEQ ID NO: 143, SEQ IDNO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148,SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ IDNO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 156 or asequence at least 95% identical to the sequence of said insert.
 22. Thenucleic acid of claim 1, wherein said nucleic acid comprises anucleotide sequence encoding a polypeptide at least 95% identical to thepolypeptide encoded by the insert which encodes a modified HIV Envglycoprotein in a sequence selected from the group consisting of SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 50, SEQID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 954, SEQ ID NO: 55,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94,SEQ ID NO: 96, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119,SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ IDNO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 142, SEQ ID NO: SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ IDNO:
 156. 23. The nucleic acid of claim 1 further comprising a backbone,wherein said backbone is a plasmid vector.
 24. The nucleic acid of claim1 further comprising a backbone, wherein said backbone is an adenoviralvector.
 25. A nucleic acid which is at least 95% identical to thenucleic acid of claim 1, wherein said nucleic acid encodes a modifiedHIV Env glycoprotein with a termination codon at position
 704. 26. Amodified HIV Env glycoprotein encoded by the nucleic acid molecule ofclaim
 1. 27. A vaccine composition comprising the nucleic acid moleculeof claim 1 and a pharmaceutically acceptable solution in aprophylactically effective dose.
 28. The composition of claim 27 furthercomprising an adjuvant.
 29. A method of generating an antibody or CTLresponse against native HIV Env comprising the step of administering thenucleic acid molecule of claim 1 to a host to generate said antibody orCTL response against native HIV Env.
 30. The method of claim 29, whereinsaid step is a primary immunization.
 31. The method of claim 29, whereinsaid step is a boost.
 32. The method of claim 29, wherein said step is aprimary immunization with a plasmid vector.
 33. The method of claim 29wherein said step is a primary immunization with a plasmid vectorexpressing gp145delCFI and further comprising a step which is a boostwith an adenoviral vector expressing gp140delCFI.